CN102630512A - Method for improving abiotic stress resistance of plants to disease sources, drought and high salinity - Google Patents

Abstract

The invention belongs to the technical field of plant molecular biology and genetic engineering, and specifically relates to a method for improving the abiotic stress resistance of plants to disease sources, drought and high salinity. The method disclosed by the invention comprises the step of adopting a chemical inducer, namely PBZ (probenazole), to perform induction, namely applying the chemical inducer, namely the PBZ, on the plants, wherein the application way is that a PBZ solution is sprayed; when spraying is performed, the concentration of the PBZ solution is controlled at 0.3-0.6mM; and in order to improve the effect of applying the chemical inducer, namely the PBZ, arabidopsis AtNPR1 genes or arabidopsis AtICS1 genes are introduced into plant tissues or cells.

Description

Improve the method for plant to pathogeny, arid and high salt environment stress resistance

Technical field

The invention belongs to molecular biology of plants and gene engineering technology field, be specifically related to a kind of method that improves plant to pathogeny, arid and high salt environment stress resistance.

Background technology

Along with the development of Plant Genome and multiple research means, the molecule mechanism of plant stress-resistance there has been more understanding, adverse circumstance inducement signal transduction pathway more clearly has 3 at least: the one, depend on the pathway of ABA; The 2nd, be independent of the stress-inducing expression of gene of ABA; The 3rd, partly depend on the signal pathway of ABA, like arabidopsis RD29AGene (Yamaguchi SK Et al., Proc. Natl. Acad. Sci. USA, 103,1988-1993,2006).In addition, find also now to comprise that materials such as brassinosteroid, ethene, jasmonic, salicylic acid are also playing the part of important role (Kaschani F in the plant drought salt tolerant is coerced Et al., Curr. Opin. Chem. Biol., 11,88-98,2007; Catinot J Et al., FEBS Lett., 4,473-478,2008).

Utilize some expression of gene in the suitable xenobiotics regulation and control grown in field crop; Quite crucial for agricultural production and grain-production, especially for the following several types of genes that higher using value is arranged in genetically modified plants: (1) increases the accumulation of purpose nutriment in seed, root, stem; (2) growth and development of plants in the cycle a certain period produce certain product and be collected in a certain plant tissue so that collect or/and separate; (3) start the expression that the toxin of special toxicity is arranged for a certain insect pest in the position that cause of disease is attacked; (4) improve plant and resist especially arid of multiple adverse circumstance, saline and alkaline ability; (5) growth cycle of target plant is regulated.Second can make bio-reactor have higher efficient, and the 4th can better be improved field crop and stand boisterous ability, ensures normal growth.

(1-dioxide PBZ), is the good reagent of control rice blast to allyl isothiazole probenazole for 3-allyloxy-1,2-benzisothiazole-1, is one of kind of consumption maximum in the present Japanese bactericide.PBZ itself with and metabolite biologically active not; Do not influence the growth of rice blast and to the infection ability of plant; It mainly is that the disease-resistant mechanism that system through activated plant self obtains resistance makes plant produce disease resistance (Michiaki Lwata; The Royal Society of Chemistry Pesticide Outlook, 28-31,2001).PBZ can induce dicotyledon generation systems such as arabidopsis and tobacco to obtain resistance, in monocotyledons such as paddy rice, can produce disease resistance through similar disease resistance response inducing plant.

Found at first when the present invention studies small-molecule substance PBZ that PBZ can improve the resistance of arabidopsis to multiple environment stress, and immediately this phenomenon has been launched systematic research.Morphological observation, cis element induce means such as variation, genetic chip analysis and mutant blocking-up analysis to confirm that finally PBZ induces the roughly signal pathway of raising arabidopsis to multiple environment stress resistance, for further through screening and utilizing small-molecule substance to improve the comprehensive advantage of plant in the growth life possibility being provided.

Summary of the invention

The purpose of this invention is to provide a kind of method that can improve the multiple environment stress of plant (comprising environment stresses such as pathogeny, arid and high salt) resistance.

The method of the raising multiple environment stress of plant (comprising environment stresses such as pathogeny, arid and high salt) resistance provided by the invention; Chemical inducer--allyl isothiazole is induced in employing; Promptly plant is applied chemical inducer PBZ, the mode that applies can be for spraying PBZ solution.When spraying, the PBZ solution concentration is controlled to be 0.3-0.6 mM, and preferred concentration is 0.5mM.Vegetable material is the vegetable material in later stage of nourishing and growing.

In order to strengthen the effect that chemical inducer PBZ applies, the present invention also imports arabidopsis to plant tissue or cell AtNPR1Gene or arabidopsis AtICS1Gene.

Among the present invention, described arabidopsis AtNPR1Gene, the polynucleotide sequence with SEQ ID NO:1 coded DNA sequence, the polynucleotides series of the SEQ ID NO:2 amino acid sequence of perhaps encoding.Arabidopsis AtNPR1 encoding proteins; Be protein, or the amino acid residue sequence of SEQ ID NO:2 is had the protein of being derived by sequence 2 with the identical activity of amino acid residue sequence of SEQ ID NO:2 through replacement, disappearance or the interpolation of one or several amino acid residue with SEQ ID NO:2 amino acid residue sequence.

Among the present invention, described arabidopsis AtICS1Gene, the polynucleotide sequence with SEQ ID NO:3 coded DNA sequence, the polynucleotides of the SEQ ID NO:4 amino acid sequence of perhaps encoding.Arabidopsis AtICS1 encoding proteins; Be protein, or the amino acid residue sequence of SEQ ID NO:4 is had the protein of being derived by sequence 4 with the identical activity of amino acid residue sequence of SEQ ID NO:4 through replacement, disappearance or the interpolation of one or several amino acid residue with SEQ ID NO:4 amino acid residue sequence.

Said gene of the present invention plays a significant role in the multiple environment stress resistance of plant raising plant.

The present invention also provides a kind of method that causes that in plant selected gene product expression increases, and concrete steps are following:

(1) with recombinant DNA thaumatropy plant;

(2) handle genetically modified plants with Compound P BZ;

(3) the render transgenic plant increases the expression of selected gene outcome in expectation.

Among the present invention, described plant can be paddy rice, corn etc.

Description of drawings

Fig. 1 is after drought stress is handled 16 days, and the wild type arabidopsis is the drought resistance performance under PBZ processing and water treatment respectively.

Fig. 2 handles the back rehydration at drought stress, and the wild type arabidopsis is the survival under PBZ processing and water treatment respectively.

Fig. 3 is under 300mM NaCl handles, and the wild type arabidopsis is the salt-resistance performance under PBZ processing and water treatment respectively.

Fig. 4 is after drought stress was handled 18 days, the wild type arabidopsis with AtNPR1Transgenic arabidopsis is the drought resistance performance under PBZ processing and water treatment respectively.Wherein, (a) the wild type arabidopsis material handled of PBZ; (b) the wild type arabidopsis material of water treatment; (c) commentaries on classics of PBZ processing NPR1The arabidopsis material of gene; (d) commentaries on classics of water treatment NPR1The arabidopsis material of gene.

Fig. 5 is after drought stress is handled 18 days, AtICS1Transgenic arabidopsis is the drought resistance performance under PBZ processing and water treatment respectively.

Fig. 6 is after drought stress is handled 13 days, and the wild type corn is the drought resistance performance under PBZ processing and water treatment respectively.

Fig. 7 is after drought stress is handled 13 days, and the wild type corn is the relative water content situation under PBZ processing and water treatment respectively.

Fig. 8 is after drought stress is handled 13 days, and the wild type corn is the MDA accumulation under PBZ processing and water treatment respectively.

Fig. 9 is under drought stress is handled, and the wild type paddy rice is the drought resistance performance under PBZ processing and water treatment respectively.

Embodiment

Embodiment 1, arabidopsis AtNPR1With AtICS1The acquisition of encoding gene.

1.1 RNA extracts

Get the about 0.1g of Arabidopsis leaf.After liquid nitrogen fully grinds, transfer to the 1.5ml centrifuge tube, add 1ml TRIzol ^R(invitrogen company), behind the mixing, room temperature was placed 15 minutes, added the 0.2ml chloroform: isoamyl alcohol (24:1), acutely shake after 15 seconds room temperature and placed 5 minutes, 13000rpm, 4 ℃ are centrifugal 15 minutes.Get supernatant and add the equal-volume isopropyl alcohol, careful mixing, room temperature was placed 15 minutes, 13000rpm, 4 ℃ are centrifugal 15 minutes.70% washing with alcohol deposition, drying at room temperature 15 minutes.Be dissolved in the ddH2O water that an amount of warp 0.1% DEPC handled, be stored in-80 ℃ subsequent use.

1.2 cDNA first chain is synthetic and reverse transcription PCR

Adopt the cDNA first chain synthetic agent box of Shen, Shanghai ability lottery industry biotech company (SHBC), total RNA reverse transcription is become cDNA according to operating guidance.Reaction system and reaction condition are respectively: total RNA of 2ug preparation; 0.5ul Rnase inhibitor adds deionized water that DEPC handled to 8.5ul, 65 ℃ of 5min of the Oligo of 2ul (dT) 18 primer.; Room temperature is placed 10min, the centrifugal 5s of 13000rpm.Add 4 μ l, 5 * First-Strand buffer more successively, 0.5 μ l RNase Inhibitor, 2 μ l 100mM DTT, 2 μ l dNTP, 1 μ l MMLV Reverse Transcriptase.Careful mixing; 37 ℃ of reverse transcriptions 1 hour, 90 ℃ 5 minutes; Cooled on ice; 13000rpm of short duration centrifugal 5 seconds, deposit in-20 ℃ for use.

1.3 AtNPR1With AtICS1The acquisition of complete sequence

According to the sequence that the arabidopsis database provides, SEQ ID NO:5 and SEQ ID NO:6 have been designed as the clone AtNPR1The primer of the PCR reaction of complete sequence, SEQ ID NO:7 and SEQ ID NO:8 are as the clone AtICS1The primer of the PCR reaction of complete sequence.The PCR reaction system is 50ul, and reaction condition is: 94 ℃ of preparatory sex change 5min, and 94 ℃ of sex change 40s, 58 ℃ of renaturation 40s, 72 ℃ are extended 60s, circulate 35 times.72 ℃ are fully extended 10min.The PCR product of gained is separated through 1% agarose gel electrophoresis.After reclaiming and being cloned into TAKARA pMD-19-T carrier,, obtain by the order-checking of Shanghai Ying Jun company through TA AtNPR1With AtICS1Sequence.

Embodiment 2, arabidopsis AtNPR1With AtICS1Transgenic analysis.

2.1 LBA4404 Agrobacterium competent cell preparation

1) contains rifamycin 40ug/ml, ruling on the YEB solid culture medium of streptomycin 100ug/ml, cultivating 48h-72h for 28 ℃;

2) choose single bacterium colony to containing rifamycin 40ug/ml, 28 ℃ are cultured to OD600 0.5 in the YEB liquid nutrient medium of streptomycin 100ug/ml;

3) cooled on ice bacterium liquid, 5000rpm, 4 ℃ of 10 minutes collection thalline;

4) 1mM Hepes pH 7.0 washings are 3 times, again with the washing of 10% glycerine once;

5) the suspension thalline divides to install in the 1.5ml centrifuge tube every pipe 40ul in 3ml 10% glycerine.

2.2 Agrobacterium-mediated Transformation

1) 200ng DNA adds and carries out electricity by following condition behind the 40ul Agrobacterium competent cell mixing and transform;

U 1.8 KV

R 200 Ω

C 25 uF

2) 800ul SOC liquid nutrient medium is added in the electric shock back, cultivates 1h for 28 ℃;

3) 4000rpm collected thalline in 10 minutes, was suspended among the 200ul SOC, was coated in to contain 100 ug/ml spectinomycins, and rifampin 40ug/ml on the streptomycin 100ug/ml LB solid culture medium, is inverted for 28 ℃ and cultivates 48h-72h.

2.3 agriculture bacillus mediated arabidopsis transforms

Arabidopsis matrix is cultivated:

Matrix components: vermiculite: black earth: perlite=9: 3: 0.5

Nutrient solution prescription:

After matrix is soaked into nutrient solution, planting seed in earthen bowl, is covered with preservative film, place under 4 ℃ of dark conditions, change (16h L/8h D) illumination after 2 days over to, cultivate under 23 ℃ of conditions.Arabidopsis grows into bolting and blooms and can supply to transform.

2.4 Agrobacterium is prepared

1) inoculation carry the purpose expression vector Agrobacterium to containing in an amount of antibiotic LB medium, 28 ℃, 220rpm shakes bacterium and is cultured to OD600 1.2;

2) 5000rpm, 4 ℃ of 10 minutes centrifugal collection thalline;

3) thalline is suspended in 5% the sucrose solution again, and transfers to OD600 0.8;

4) add Silwet L-77 to final concentration be 0.03%.

2.5 arabidopsis transforms

Get the arabidopsis material, be inverted and soak acrial part in ready Agrobacterium solution, rocked about 3 seconds, take out, be placed under the concealment condition, the 24h that preserves moisture changes normal condition over to and cultivates.

2.6 arabidopsis transformant screening

Collect the seed that transforms the back arabidopsis.Seed is with 0.01% HgCl2 surface sterilizing 8 minutes, and aseptic water washing 4 times is suspended in 0.1% the agarose, by 2000 seeds of every flat board (diameter 15cm) (about 40mg), is layered on the 1/2 MS medium of kanamycin 50mg/L then.Flat board was placed 4 ℃ of dark refrigerators 2 days, transfer to (16h L/8h D) illumination, cultivate under 23 ℃ of conditions.Can screen the transfer-gen plant of providing kalamycin resistance in about about 10 days.The plant of tool resistance is transferred to matrix cultivate, and results T2 is for seed.

Embodiment 3, arabidopsis material receive PBZ to induce the back to improve the resistance to the drought stress adverse circumstance.

Under the drought stress treatment conditions, PBZ handled after 16 days, and the obvious growth discomfort does not appear in wild type arabidopsis material, and the phenomenon of significantly wilting has appearred in the wild type arabidopsis material of water treatment this moment.After handling 17 days, carry out the rehydration operation, the wild type arabidopsis material that PBZ handles can normally be survived, and the wild type arabidopsis material of water treatment this moment can not be survived.It is more obvious that this type phenomenon shows in genetically modified plants, and facilitation effect is more remarkable, and it mainly shows as after PBZ induces processing, occurred the phenomenon of wilting in late at least 2 days than wild type material.

Experimental example 4, corn, rice material receive PBZ to induce the back to improve the resistance to the drought stress adverse circumstance.

PBZ induces processing can significantly improve corn, the rice material resistance to the drought stress adverse circumstance.The accumulation of relative water content and MDA can explain well that all PBZ induces the enhancing of back drought resistance.

< 110>Fudan University

< 120>improve the method for plant to pathogeny, arid and high salt environment stress resistance

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<170> PatentIn version 3.3

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Leu Val Ser Val Ala Gly Ile Gly Ser Ala Val Phe Phe Arg Asp Leu

165 170 175

Asp Pro Phe Ser His Asp Asp Trp Arg Ser Ile Arg Arg Phe Leu Ser

180 185 190

Ser Thr Ser Pro Leu Ile Arg Ala Tyr Gly Gly Met Arg Phe Asp Pro

195 200 205

Asn Gly Lys Ile Ala Val Glu Trp Glu Pro Phe Gly Ala Phe Tyr Phe

210 215 220

Ser Val Pro Gln Val Glu Phe Asn Glu Phe Gly Gly Ser Ser Met Leu

225 230 235 240

Ala Ala Thr Ile Ala Trp Asp Asp Glu Leu Ser Trp Thr Leu Glu Asn

245 250 255

Ala Ile Glu Ala Leu Gln Glu Thr Met Leu Gln Val Ser Ser Val Val

260 265 270

Met Lys Leu Arg Asn Arg Ser Leu Gly Val Ser Val Leu Ser Lys Asn

275 280 285

His Val Pro Thr Lys Gly Ala Tyr Phe Pro Ala Val Glu Lys Ala Leu

290 295 300

Glu Met Ile Asn Gln Lys Ser Ser Pro Leu Asn Lys Val Val Leu Ala

305 310 315 320

Arg Asn Ser Arg Ile Ile Thr Asp Thr Asp Ile Asp Pro Ile Ala Trp

325 330 335

Leu Ala Gln Leu Gln Arg Glu Gly His Asp Ala Tyr Gln Phe Cys Leu

340 345 350

Gln Pro Pro Gly Ala Pro Ala Phe Ile Gly Asn Thr Pro Glu Arg Leu

355 360 365

Phe Gln Arg Thr Gln Leu Gly Val Cys Ser Glu Ala Leu Ala Ala Thr

370 375 380

Arg Pro Arg Ala Ala Ser Ser Ala Arg Asp Met Glu Ile Glu Arg Asp

385 390 395 400

Leu Leu Thr Ser Pro Lys Asp Asp Leu Glu Phe Ser Ile Val Arg Glu

405 410 415

Asn Ile Arg Glu Lys Leu Asn Gly Ile Cys Asp Arg Val Val Val Lys

420 425 430

Pro Gln Lys Thr Val Arg Lys Leu Ala Arg Val Gln His Leu Tyr Ser

435 440 445

Gln Leu Ala Gly Arg Leu Thr Lys Glu Asp Asp Glu Tyr Lys Ile Leu

450 455 460

Ala Ala Leu His Pro Thr Pro Ala Val Cys Gly Leu Pro Ala Glu Glu

465 470 475 480

Ala Arg Leu Leu Ile Lys Glu Ile Glu Ser Phe Asp Arg Gly Met Tyr

485 490 495

Ala Gly Pro Ile Gly Phe Phe Gly Gly Glu Glu Ser Glu Phe Ala Val

500 505 510

Gly Ile Arg Ser Ala Leu Val Glu Lys Gly Leu Gly Ala Leu Ile Tyr

515 520 525

Ala Gly Thr Gly Ile Val Ala Gly Ser Asp Pro Ser Ser Glu Trp Asn

530 535 540

Glu Leu Asp Leu Lys Ile Ser Gln Val Arg Ala Phe Val Gln Lys Met

545 550 555 560

Phe Ser Asp Ile Met Val Leu Cys Tyr Gln Asn Pro Asn Phe Tyr Ser

565 570 575

Leu Phe Cys Cys Cys Phe Cys Ser Ser Pro Ser Gln Leu Asn Met Lys

580 585 590

Gln Gln His Leu Tyr Arg Arg Leu Ile Glu Glu Arg Val Thr Phe Val

595 600 605

Phe Asp Cys Phe Val Cys Met Gly Asp Lys Gly Phe Ser Gln

610 615 620

<210> 5

<211> 30

<212> DNA

< 213>description of artificial sequence: primer

<400> 5

agttgataag gtctcttcgt tgattagcag 30

<210> 6

<211> 30

<212> DNA

< 213>description of artificial sequence: primer

<400> 6

ggtacagcaa aaattacact aagaggcaag 30

<210> 7

<211> 33

<212> DNA

< 213>description of artificial sequence: primer

<400> 7

attggtacca tggcttcact tcaattttct tct 33

<210> 8

<211> 31

<212> DNA

< 213>description of artificial sequence: primer

<400> 8

atatctagat tattgtgaga accccttatc c 31

Claims (5)

1. method that improves plant pathogeny, arid and high salt environment stress resistance is characterized in that adopting that chemical inducer--allyl isothiazole is induced, and promptly plant is applied chemical inducer PBZ, and the mode that applies is for spraying PBZ solution; When spraying, the PBZ solution concentration is controlled to be 0.3-0.6 mM.

2. the method for raising plant pathogeny according to claim 1, arid and high salt environment stress resistance is characterized in that also plant tissue or cell being imported arabidopsis AtNPR1Gene or arabidopsis AtICS1Gene;

Described arabidopsis AtNPR1Gene, the polynucleotide sequence with SEQ ID NO:1 coded DNA sequence, the polynucleotides series of the SEQ ID NO:2 amino acid sequence of perhaps encoding;

Described arabidopsis AtICS1Gene, the polynucleotide sequence with SEQ ID NO:3 coded DNA sequence, the polynucleotides of the SEQ ID NO:4 amino acid sequence of perhaps encoding.

3. method that causes that in plant selected gene product expression increases is characterized in that concrete steps are following:

(1) with recombinant DNA thaumatropy plant;

(2) handle genetically modified plants with Compound P BZ;

(3) the render transgenic plant increases the expression of selected gene outcome in expectation.

4. method according to claim 1 and 2 is characterized in that described plant is paddy rice or corn.

5. method according to claim 3 is characterized in that described plant is paddy rice or corn.

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