CN104170823A - Small molecule compound for enhancing plant stress resistance - Google Patents
Small molecule compound for enhancing plant stress resistance Download PDFInfo
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Abstract
The invention relates to a small molecular compound for enhancing plant stress resistance, and firstly reveals that small molecular compound AM1 has similar physiological activity with ABA, can significantly enhance plant tolerance to abiotic stress (commonly including cold, drought, salinity, osmotic pressure, heat and the like) widely existing in environment, and has higher application value.
Description
Technical field
The invention belongs to biotechnology and botany field; More specifically, the present invention relates to a kind of micromolecular compound that strengthens stress resistance of plant.
Background technology
Abscisic acid (Abscisic Acid, ABA) be balance plant endogenous hormones and the key factor about growth activity metabolism, there is the ability that promotes plant Balance Absorption water, fertilizer and coordinate internal metabolism, root that can Effective Regulation plant/be preced with and nourish and grow and reproductive growth, has important function to the quality, the output that improve crops.By using ABA, can reduce the amount of application of chemical pesticide, there are important physiological activity and using value aspect many improving quality of agricultural product etc.In addition, Exogenous ABA can cause closing rapidly of Stoma of Leaves, suppresses transpiration, can be used for the fresh-keeping of flower, or in the transportation of crop seedling implanting and cultivating, prevents from wilting; ABA can also control flower bud differentiation, regulates the florescence, has very large using value on flower gardening.ABA belongs to the plant growth regulator of pure natural, can improve growth quality and ripening rate and the quality of crop in the bad growing environments such as low temperature, arid, cold spell in spring, salt marsh, damage by disease and insect, improves the per unit area yield output of middle-and-low-yielding fields, reduces chemical pesticide consumption.ABA also can be widely used in the Urban Greening such as urban lawn, gardens, is applied to water-saving agriculture, the industrialized agriculture in west area, and the restoration and reconstruction of ecological vegetation are significant for developing china agriculture industrialization.But current bottleneck is that simple (+) with natural activity-ABA is unstable, manually synthetic difficulty is larger, and production cost is high.Due to the difference in expensive price and activity, ABA is not widely used in agricultural production always, and various countries scientist is finding the cheap method of producing of natural type ABA.
Through the ABA signal research of many decades, protein phosphatase 2C (Protein Phosphatases2C, PP2Cs) and SNF1 associated kinase subfamily 2 (Subfamily2of the SNF1-related kinases, SnRK2kinases) be confirmed as ABA acceptor downstream signal hinge.The START protein family that a nearest class comprises 14 members is confirmed as ABA acceptor, called after PYR1 (Pyrabactin Resistance1)/class PYR1 (PYR1-like, PYL) acceptor (for for simplicity, being designated hereinafter simply as PYL acceptor).ABA has strengthened the latter in conjunction with PYL acceptor and some specific PP2C phosphoprotein phosphatase (comprises ABI1, ABI2 and HAB1 etc.) binding ability, suppressed the activity of these PP2C phosphoprotein phosphatases simultaneously, then activate the SnRK2 protein kinase in downstream, thereby activated follow-up stress reaction.Nearest structural research shows that PYL acceptor and PP2C phosphoprotein phosphatase have formed the co-receptor of ABA, and has determined and the conservative mechanism of ABA perception and signal correction.
Park, S.Y.et al.<Abscisic acid inhibits type2C protein phosphatases via the PYR/PYL family of START proteins>Science324, 1068-71 (2009) mentions micromolecular compound pyrabactin and has the function of similar ABA, but its physiologically active will a low order of magnitude compared with ABA, and can only be in conjunction with a few PYL acceptor, mainly play the effect of ABA analog in the seed germination stage, can not strengthen the drought resistance in plant seedling stage and maturing stage, using value in agricultural production is low.
Therefore, this area is necessary further to develop ABA substitute products, once find such micromolecular compound, its economic benefit producing, social benefit and Environmental Effect Yidu will be very remarkable.
Summary of the invention
The object of the present invention is to provide cheapness and possess the active natural abscisic acid substitute of abscisic acid (Abscisic Acid, ABA).
In a first aspect of the present invention, the purposes of a kind of formula (I) compound or its salt is provided, for strengthening stress resistance of plant or the Agrotechnical formulation for the preparation of enhancing stress resistance of plant,
In another preference, described resistance is the abiotic stress resistance that ABA participates in.
In another preference, the abiotic stress resistance that described ABA participates in includes but not limited to: drought resistance, cold resistance, Salt And Alkali Tolerance or resistance to osmotic pressure etc.
In another preference, described plant comprises: the plant that contains the ABA of PYR/PYL family acceptor.
In another preference, described plant comprises: economic crops and cereal crops.
In another preference, described plant includes but not limited to land plant.
In another preference, described plant includes but not limited to: crucifer (comprises that mouse ear mustard belongs to or rape belongs to, as arabidopsis, Chinese cabbage, a variety of Chinese cabbage), plant of Solanaceae (comprising Nicotiana, as tobacco), gramineous plants (comprising Oryza, as paddy rice).
In another preference, described plant includes but not limited to: arabidopsis, Chinese cabbage, rape, tobacco, tomato, capsicum, wheat, paddy rice, barley, corn, Chinese sorghum, oat, rye, sugarcane, cotton, soybean, beet, sunflower etc.
In another preference, described formula (I) compound or its salt also for: promote the interaction of ABA acceptor PYL albumen and multiple PP2C phosphoprotein phosphatases; Or weaken the transpiration of blade.
In another aspect of this invention, provide a kind of method that strengthens stress resistance of plant, use formula (I) compound or its salt to plant:
In a preference, use formula (I) compound or its salt of effective dose to plant.
In another preference, described effective dose is 1-500 μ M; Be preferably 5-200 μ M; Be more preferably 20-100 μ M;
In another preference, described resistance is the abiotic stress resistance that ABA participates in.
In another preference, the abiotic stress resistance that described ABA participates in be drought resistance, cold resistance, Salt And Alkali Tolerance, resistance to osmotic pressure or stable on heating at least one.
In another preference, the plant of described plant for containing the ABA of PYR/PYL family (abscisic acid) acceptor.
In another preference, described plant includes but not limited to: crucifer, plant of Solanaceae, gramineous plants.
In another preference, described plant includes but not limited to: arabidopsis, tobacco, Chinese cabbage, rape, tomato, capsicum, wheat, paddy rice, barley, corn, Chinese sorghum, oat, rye, sugarcane, cotton, soybean, beet, sunflower.
In another aspect of this invention, provide a kind of Agrotechnical formulation, described Agrotechnical formulation comprises: formula (I) compound or its salt of effective dose; And the upper acceptable carrier of agricultural;
In a preference, in described Agrotechnical formulation, the content of formula (I) compound or its salt in Agrotechnical formulation is 1-500 μ M; Be preferably 5-200 μ M; Be more preferably 20-100 μ M; And/or
In another preference, described Agrotechnical formulation is concentrate formulation, and the content of its Chinese style (I) compound is 1-1000mM; Be preferably 10-500mM, as 50mmM, 100mM, 200mM, 400mM.
In described agricultural, acceptable carrier includes, but is not limited to: and surfactant (as Tween-20, Silwet-77, preferably content is 0.02% (v/v)).
Other side of the present invention, due to disclosure herein, is apparent to those skilled in the art.
Brief description of the drawings
Fig. 1, AM1 are the PYL receptor stimulating agents of a broad-spectrum high efficacy.
The two-dimensional structure formula of a, AM1 and ABA.
B, phosphoprotein phosphatase HAB1 activity experiment.AM1 under ABA working concentration can, by promoting the combination of multiple PYL acceptors (PYR1, PYL1, PYL2, PYL3, PYL5, PYL7) and phosphoprotein phosphatase HAB1, significantly suppress the latter's phosphatase activity.
The interaction of c-e, AlphaScreen experimental verification PYL acceptor and phosphoprotein phosphatase HAB1.Numerical value in the form of figure below is EC50 value.
Fig. 2, yeast two-hybrid experiment confirm that AM1 can promote the interaction of PYL receptor family and multiple PP2C phosphoprotein phosphatases.
A, yeast are not containing the medium growing state of isoleucine and tryptophan (Leu/-Trp).
B, yeast are not containing isoleucine, growing state in the DMSO control medium of tryptophan and histidine (Leu/-Trp/-His).
C, yeast are containing 2 μ M ABA and are not containing isoleucine, growing state on the medium of tryptophan and histidine (Leu/-Trp/-His).
D, yeast are containing 2 μ M AM1 and are not containing isoleucine, growing state on the medium of tryptophan and histidine (Leu/-Trp/-His).
The crystal structure of Fig. 3, PYL2-AM1-HAB1 compound.
The three-dimensional structure schematic diagram of a, compound.
The two-dimensional structure partial schematic diagram of b, compound.
Fig. 4, AM1 process the expression of coercing related gene in can inducing plant body.
In a, wild type arabidopsis plant in seedling stage, coerce related gene and all raise after the ABA of 50 μ M and AM1 process, and in PYL acceptor three mutant strains, level-off significantly reduces.
Compared with the control group that b, RNA-seq result show to process with DMSO, in the sample sets that ABA and AM1 process, there are respectively 4529 and 4454 genes to have differential expression, wherein there are 3644 genes all to have differential expression in the sample sets of ABA and AM1 processing, account for respectively both 80.5% and 81.8%.
The most significant relevant gene of plant responding arid, cold, osmotic pressure and salt stress just of difference in c, above-mentioned 3644 genes.
Fig. 5, AM1 can significantly suppress seed germination.
A) PYL acceptor three mutant strain (pyr1; Pyl1; Pyl4) sprout the photo after 4 days.All PYL acceptor three mutant strains are all opened cotyledon, and in DMSO control group, wild type plant is all opened cotyledon, and only grow radicle containing most wild type plant in the experimental group of 1 μ M ABA and AM1, and sprouting is significantly inhibited.
B) PYL acceptor three mutant strain (pyr1; Pyl1; Pyl4) sprout the germination rate statistics after 4 days, open as sprouting standard using cotyledon.
Fig. 6, AM1 strengthen the drought resistance of plant by reducing transpiration.
The experiment of a, soil drought, two weeks large wild type arabidopsiss stop feeding water and on blade, sprayed AM1 or ABA solution every 1 day.After 2 weeks, the large portion of plant that sprays DMSO contrast solution is withered and recover cannot restore after feedwater, and the plant that sprays AM1 or ABA still keeps green, recovers feedwater and restores rapidly afterwards for one day.
B, arid wild type arabidopsis survival rate after 2 weeks, calculates according to recovering the survival rate of feedwater after 1 day.
C, the in vitro percentage of water loss of the large wild type plant leaf of surrounding.
Embodiment
The present invention has disclosed one and has possessed abscisic acid (Abscisic Acid, ABA) active natural abscisic acid substitute--abscisic acid analogs 1 (ABA mimic1, AM1), micromolecular compound AM1 of the present invention possesses the similar physiologically active with ABA, can be in conjunction with most of PYL acceptor, sprouting and vegetative stage all plays the effect of ABA analog, the resistance that can significantly strengthen plant is (as drought resistance, cold resistance etc.), because most cereal crops and economic crops all contain the ABA of PYR/PYL family acceptor, this compound has higher using value.The invention solves that natural A BA chemical stability is low and production cost is high, be difficult to be applied to the defect of agricultural production.
Compound and uses thereof
First the present invention provides a kind of compound as shown in structural formula (I):
The present invention also comprises isomer, racemic modification, salt, hydrate or the precursor of above-claimed cpd, as long as they also have the effect of enhancing stress resistance of plant (preferably for abiotic stress resistance, as drought resistance).Described " salt " refers to that described compound reacts the salt generating with inorganic acid, Organic Acid and Base metal or alkaline earth metal etc.These salt include, but is not limited to: (1) and the salt that inorganic acid forms as follows: example hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid; (2) with the salt that organic acid forms as follows, as acetic acid, oxalic acid, succinic acid, tartaric acid, methanesulfonic acid, maleic acid or arginine.Other salt comprises the salt forming with alkali metal or alkaline earth metal (as sodium, potassium, calcium or magnesium), with ester, carbamate, or the form of " precursor " of other routine.Compound has one or more asymmetric centers.So these compounds can be used as racemic mixture, independent enantiomter, independent diastereoisomer, non-enantiomer mixture, cis or transisomer existence.
Described " biology is coerced " refers to the impact of coercing being applied by some biologies (such as insect pest, germ).Described " abiotic stress " is the environmental influence being caused by abiotic, as: arid, cold, saline and alkaline, osmotic pressure, sweltering heat, flood, mineral deficiency and disadvantageous PH etc.
Described " precursor of compound " refers to be applied to after plant, the precursor of this compound carries out metabolism or chemical reaction and is transformed into a kind of compound of structural formula (I) in plant corpus, or a compound of chemical structural formula (I) salt or the solution that form.
Those skilled in the art should understand, after obtaining the structure of cicada the compounds of this invention, can be by multiple method well known in the art, utilize known raw material, obtain compound of the present invention, the method of extracting such as chemosynthesis or from biological (as animal or plant), these methods all comprise in the present invention.
Synthetic chemistry transformation, protection functional group methodology (protect or go and protect) are helpful to synthetic application compound, and be technology commonly known in the art, as R.Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T.W.Greene and P.G.M.Wuts, Protective Groups in Organic Synthesis, 3
rded., John Wiley and Sons (1999); L.Fieser and M.Fieser, Fieser and Fieser ' s Reagents for Organic Synthesis, John Wiley and Sons (1994); And L.Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, has open in John Wiley and Sons (1995).
A kind of method of synthesis type (I) compound is as follows:
Based on the inventor's new discovery, the invention provides the purposes of the compound shown in formula (I) or its isomer, racemic modification, salt, hydrate or precursor, they are by acting on ABA acceptor, should there is the purposes similar with ABA, preferably, can be used for strengthening stress resistance of plant (if drought resistance, cold resistant, Salt And Alkali Tolerance, resistance to osmotic pressure are or/and warm tolerance); Or for the preparation of drought-enduring, cold resistant, Salt And Alkali Tolerance, resistance to osmotic pressure or/and the plant that temperature capacity strengthens.
In the present invention, have no particular limits for being applicable to plant of the present invention (or crop), as long as it has the ABA of PYR/PYL family acceptor and downstream signal path thereof, as various crops, flower plant or forestry plant etc.Described plant is such as being (being not limited to): dicotyledon, monocotyledon or gymnosperm.More specifically, described plant includes, but is not limited to: wheat, barley, rye, paddy rice, corn, jowar, beet, apple, pears, Lee, peach, apricot, cherry, strawberry, rasp berry, blackberry, blueberry, beans, French beans, pea, soybean, rape, mustard, opium poppy, olive, sunflower, coconut, castor oil plant, cocoa bean, peanut, cucurbit, cucumber, watermelon, cotton, flax, hemp, jute, citrus, lemon, grapefruit, spinach, piemarker lettuce, asparagus, cabbage, Chinese cabbage, a variety of Chinese cabbage, carrot, onion, potato, tomato, green pepper, avocado, cassia bark, camphor, tobacco leaf, nut, coffee, eggplant, sugarcane, tealeaves, pepper, vine, oyster fiber crops grass, banana, natural rubber tree and ornamental plants etc.
As a kind of optimal way, described " plant " includes but not limited to:: crucifer (comprises that mouse ear mustard belongs to or rape belongs to, as arabidopsis, Chinese cabbage, a variety of Chinese cabbage), plant of Solanaceae (comprising Nicotiana, as tobacco), gramineous plants (comprising Oryza, as paddy rice).
Agrotechnical formulation
As used herein, normally agricultural plant growth regulator of term " Agrotechnical formulation of the present invention ", it contains formula (I) compound or its isomer, racemic modification, salt, hydrate or precursor as the active component that strengthens stress resistance of plant (as drought resistance); And the upper acceptable carrier of agricultural or excipient.
In the present invention, term " contains " and represents that various compositions can be applied in mixture of the present invention or composition together.Therefore, term " mainly by ... composition " and " by ... form " be included in during term " contains ".
In the present invention, " acceptable in agricultural " composition is to be applicable to plant and without material excessive bad side reaction (as toxicity, stimulation and allergy), that have rational benefit/risk ratio.
In the present invention, " acceptable carrier in agricultural " is acceptable solvent, suspending agent or excipient in the Pesticide Science for formula of the present invention (I) compound or its isomer, racemic modification, salt, hydrate or precursor being sent to plant.Carrier can be liquid or solid.Be applicable to acceptable carrier in agricultural of the present invention and comprise (but being not limited to): water, buffer solution, DMSO, surfactant are as Tween-20 and combination thereof.In any agricultural well known by persons skilled in the art, applicable carrier is acceptable, and can be used in the present invention.Optionally, described preparation also can comprise at least one surfactant, weed killer herbicide, bactericide, insecticide or fertilizer.
The present invention also provides the method for preparing described Agrotechnical formulation, comprises the compound shown in use formula (I).Upper to the formula of effective dose (I) compound and agricultural acceptable carrier can be mixed and obtains Agrotechnical formulation of the present invention, the content of active component in composition can be for example 1-500 μ M; Be preferably 5-200 μ M; Be more preferably 20-100 μ M.Described Agrotechnical formulation can be also concentrate formulation, and the content of its Chinese style (I) compound is 1-1000mM; Be preferably 10-500mM, as 50mM, 100mM, 200mM, 400mM.
The formulation of Agrotechnical formulation of the present invention can be diversified, as long as the formulation that can make active component effectively arrive in plant corpus is all fine.From the position that is easy to preparation and uses, preferred Agrotechnical formulation is a kind of spray or pharmaceutical solutions.
The present invention also provides the method for a kind of enhancing stress resistance of plant (as drought resistance), comprises step: use formula (I) compound or its isomer, racemic modification, salt, hydrate or precursor to plant.The amount of application of active component is effective dose.
Use and can adopt known the whole bag of tricks, for example, by spraying on plant leaf blade, propagating materials, spray, dust or sowing said composition, or brush or splash or otherwise make plant contact composition, if seed, by being coated with, wrapping up or otherwise process seed.The another kind of method of directly processing plant or seed before plantation, also can be by the medium of preparation introducing soil of the present invention or other seed to be sowed.In some embodiments, also can use carrier.Described carrier can be as above solid-state, liquid.
Major advantage of the present invention is:
The contained micromolecular compound of the present invention can be in plant corpus the accurately effect of simulating plant hormone abscisic acid (Abscidic acid, ABA), significantly strengthen the resistance (as drought resistance, cold resistance etc.) of plant.Advantage is as follows:
1) consumption is few.The present invention only need run into arid in the situation that and use plant, does not need that whole growth cycle is omnidistance to be used;
2) easy to use.Water soluble under working concentration, can directly use existing agricultural spraying device;
3) selectivity is strong, and side effect is little.The resistance (as drought resistance, cold resistance) that sprays rear main enhancing plant, does not affect for other physiological functions of plant;
4) with respect to abscisic acid, chemosynthesis of the present invention is easy, and cost is low; With
5) do not relate to any transgenic technology.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, writes molecular cloning experiment guide, the third edition, Science Press, the condition described in 2002, or the condition of advising according to manufacturer conventionally as J. Pehanorm Brooker etc. according to normal condition.
I. materials and methods
Plant growth
The model plant arabidopsis (Arabidopsis thaliana) using in experiment comprises that Colombia (Col-0) is environmental and based on the ecotypic PYL Trimutant of Col-0 pyr; Pyl1; Pyl4.Tobacco practical in experiment is Ben Shi cigarette (Nicotiana benthamiana).
Arabidopsis growth temperature is 22 DEG C, and tobacco growing temperature is 24 DEG C, and light intensity is 75 μ molm
– 2s
– 1, the photoperiod comprises two kinds of long-day and short-day, and the former is 16 hours illumination/8 hour dark, and the latter is 8 hours illumination/16 hour dark.
As indicated without special, the plant growth culture medium using in experiment is 1/2MS (the Murashige and Skoog) solid culture medium containing 1% (w/v) sucrose and 0.6% (w/v) agar.
The compound using in experiment, purchased from Life Chemical Inc, uses DMSO to dissolve, and storage concentration is 100mM, is diluted with water to respective concentration when use.
Protein expression and purification
With the arabidopsis gene PYL1 (amino acid sequence 36-211) of 6 × His and the two sequence labels of SUMO, the construction method of the recombinant plasmid of PYL2 (amino acid sequence 14-188) and the arabidopsis gene HAB1 with Biotin sequence label (amino acid sequence 172-511) refers to " A gate-latch-lock mechanism for hormone signalling by abscisic acid receptors " (<Nature>Vol462, 3December2009), with other arabidopsiss PYL gene of 6 × His and the two sequence labels of SUMO, comprise PYR1, PYL3, PYL4, PYL5, PYL6, PYL7, PYL8, PYL9 is identical with PYL1/PYL2 with the construction method of the recombinant plasmid of PYL10 (above 9 genes are full gene coded sequence).
Above-mentioned recombinant plasmid is proceeded to competent cell BL21 (DE3), be inoculated into the 200ml LB liquid nutrient medium that contains Amp resistance, 37 DEG C of 200rpm overnight incubation; Be seeded in the LB liquid nutrient medium that 2L contains Amp resistance and expand cultivation by 1:50-1:100,37 DEG C of 200rpm cultivate 3-4 hour, and 16 DEG C of low temperature are cultured to OD
600=0.8-1.0 left and right.Induce and spend the night with 100 μ M IPTG with the two PYR1 of sequence label of 6 × His and SUMO or the recombinant plasmid of PYL1 or PYL2, and HAB1 recombinant plasmid with Biotin sequence label is induced with 100 μ M IPTG and 40 μ M biotin simultaneously.
Bacterium liquid centrifugal collection thalline in low speed large capacity centrifuge by induction after 16 hours, 4 DEG C with the centrifugal 20min of 4000rpm rotating speed.The resuspended thalline of 50ml Extraction buffer for every 2L bacterium liquid (containing 20mM Tris, pH8.0,200mM NaCl and 10% (v/v) glycerine), then carries out 1000Pa pressure breaking 3-5 time at 4 DEG C.Cell after fragmentation carries out ultracentrifugation, and the centrifugal 30min of 16000rpm repeats 2 times, collects supernatant and crosses affinity column.
For the PYR/PYL albumen with 6 × His and the two sequence labels of SUMO, select 50ml affinity chromatography Ni post (50ml Ni-NTA column, purchased from GE company); First use 10% buffer B (containing 20mM Tris, pH8.0,200mM NaCl, 500mM imidazole and 10% glycerine) balance 600ml, then use 200ml50% buffer B wash-out, finally use 100ml100% buffer B wash-out; The albumen of resolving for crystal and ulp1 enzyme carry out enzyme with the mixed in molar ratio of 1000:1 and cut dialysed overnight; Albumen after enzyme is cut is after an affinity chromatography Ni post; Collect elute soln for liquid (containing 25mM Tris, pH8.0,200mM ammonium acetate, 1mM dithiotreitol and 1mM EDTA) further separation and purification albumen of mistake HiLoad26/60Superdex200 solvent resistant column (purchased from GE company).
For the HAB1 albumen with Biotin sequence label, cross 50ml MBP affinity column (purchased from GE company); First use 10% buffer solution C (containing 20mM Tris, pH8.0,200mM NaCl, 10mM Maltose and 10% glycerine) balance 600ml, then use 200ml50% buffer solution C wash-out, finally use 100ml100% buffer solution C wash-out; Collect elute soln for liquid (containing 20mM Tris, pH8.0,200mM NaCl and 10% glycerine) and cross the further separation and purification albumen of HiLoad26/60Superdex200 solvent resistant column.
Albumin crystal is resolved
Before crystallization, the PYL2 after enzyme is cut and HAB1 albumen and compound, by the mixed in molar ratio of 1:1:5, are concentrated to 6mg/ml for a crystal.Adopt sessile drop method point crystal, 1 μ l complex proteins and 1 μ l well buffer mixing are cultivated as for room temperature (20 DEG C); The well buffer of PYL2-AM1-HAB1 compound crystal comprises 0.1M Succinic acid and 15%PEG3350, PYL2-AM2/AM3-HAB1 (unless otherwise indicated, "/" represents "or") the well buffer of compound crystal comprises 0.2M ammonium sulphate, 0.1M BIS-Tris, pH6.0,25%PEG3350.After one day, can see that crystal occurs, can grow to 100-120 μ m in about 3-4 days.Crystal carries out X-ray diffraction and collects diffraction data at Shanghai synchrotron radiation light source, and the data of collection are carried out data processing with HKL2000, then resolves composite structure according to the relevant PYR/PYL receptor structure model of having delivered.
AlphaScreen experiment
AlphaScreen kit is purchased from Perkin Elmer, in 100 μ l experimental systems, contain 10 × buffer (50mM MOPS, pH7.4, 50mM NaF, 50mM CHAPS, 0.1mg/ml bovine serum albumin), the HAB1 of each 100nM with Biotin sequence label and the PYR1/PYL1/PYL2 albumen with 6 × His and the two sequence labels of SUMO, 100 μ M (+)-ABA/pyrabactin/AM1/AM2, 5ug/ml donor beads and acceptor beads (Perkin Elmer), after lucifuge incubated at room 1.5 hours, be placed in Envision plate reading machine (purchased from Perkin Elmer) and carry out reading by the AlphaScreen program of setting.
The biopsy of HAB1 phosphatase is surveyed
In reaction system, contain 50mM imidazoles, pH7.2,5mM MgCl
2, 0.1% beta-mercaptoethanol, 0.5 μ gml
-1bSA, the HAB1 albumen of 100nM with Biotin sequence label, the PYL receptor protein of 500nM with the two sequence labels of 6 × His-SUMO and the compound of respective concentration, incubated at room 30 minutes, add containing 11 amino acid whose MALDI-PSDs and continue reaction 20 minutes as substrate, this MALDI-PSD is the 170-180 amino acids of SnRK2.6 protein kinase, wherein the phosphorylation serine (HSQPK of 175
psTVGTP, purchased from Jin Sirui) be known HAB1 target site.After 20 minutes, add colour reagent (purchased from BioVision), read the absorption value of 650nm wavelength by microplate reader.
Yeast two-hybrid experiment
With 14 PYL receptor protein (PYR1 of method amplification arabidopsis of PCR, PYL1-13), cut the rear multiple clone site that is cloned into respectively pGADT7 (purchased from Clontech) with corresponding restriction enzyme, four PP2C phosphatase (HAB1 increase, HAB2, ABI1, PP2CA) coding region (CDS), cut the rear multiple clone site that is cloned into respectively pBD-GAL4Cam carrier (purchased from Stratagene) with corresponding restriction enzyme.Carrier enters yeast strain AH109 (purchased from Clontech) according to corresponding PYL-PP2C combination (with reference to Fig. 2) corotation after order-checking qualification, is placed in that the SD medium (L/-T) containing Leu and Trp is not upper, cultivates 2 days for 30 DEG C.Get the yeast bacterial plaque that growth conditions is good and be diluted to OD
600=0.3, distinguish again 5 times and 25 times of dilute with waters, respectively get 1 μ l in 1) not containing the SD medium (as positive control) of Leu and Trp (L/-T), 2) not containing Leu, on Trp and His (L/-T/-H) and the SD medium containing 1mM3-AT and 2 μ M (+)-ABA/AM1/AM2 or 0.05%DMSO (as negative control), be placed at 30 DEG C and cultivate again and then take pictures for 2 days.
Table 1, PCR primer (underscore part is restriction endonuclease sites)
Gene expression analysis
(1) RNA extracting
The Col-0 Arabidopsis thaliana Seedlings of growing on 1/2MS solid culture medium 10 days is processed 6 hours with 50 μ M (+)-ABA/AM1/AM2 or 0.05%DMSO (contrast), use TRIzol reagent (purchased from Invitrogen) by specification extracted total RNA, in extractive process, add the not Dnase (purchased from Qiagen) containing Rnase and remove residual genomic DNA, the total RNA NanoDrop spectrophotometer (purchased from Thermo Scientific) obtaining carries out checking order for reverse transcription or RNA after concentration determination.
(2) reverse transcription and quantitative fluorescent PCR
Reverse transcription reaction uses TransScript RT kit (purchased from Invitrogen) and carries out according to its experimental procedure, and each sample is got the total RNA of 2 μ g and carried out reverse transcription, and the cDNA obtaining is used as the template of quantitative fluorescent PCR.Quantitative fluorescent PCR system is used SYBR Premix Ex Taq
tMiI kit (purchased from TaKaRa) also configures by its specification, uses CFX96 quantitative real time PCR Instrument (purchased from BIO-RAD) to react.Every kind of processing is got 3 biology and is repeated and carry out twice experiment repetition, and ACT7 gene is used as internal reference.
Table 2, fluorescence quantification PCR primer
(3) transcribe group order-checking and data analysis
Transcribing group order-checking uses the HiSeq2000 high-flux sequence instrument of Illumina company to complete by Mei Ji company.Every kind of processing is got 2 biology and is repeated sample presentation, and each sample exceedes 10M valid data (clean reads, average length >49bp).Use SAM to obtain 50 μ M (+)-ABA/AM1/AM2 and process the difference expression gene of processing with respect to DMSO, the expression correlation of different sample rooms is represented by Spearman correlation coefficient.
Seed germination, the in vitro dehydration experiment of soil drought and blade
(1) seed germination
The arabidopsis Col-0 ecotype and PYL acceptor three deletion mutant (pyr1; Pyl1; Pyl4) (referring to " Abscisic Acid Inhibits Type2C Protein Phosphatases via the PYR/PYL Family of START Proteins ", " Science " Vol324, seed 22May2009) is placed on 4 DEG C of refrigerator vernalization 3 days with NaClO sterilization, then sows on the 1/2MS solid culture medium containing 1 μ M (+)-ABA/AM1/AM2 or 0.05%DMSO.On the medium of each 6cm diameter, sow two strains simultaneously, 15 seeds of each strain sowing, every kind of compound is established 4 repetitions.Medium is placed in 22 DEG C of long-day cultivations, opens as the mark of sprouting using cotyledon, adds up germination rate every day.
(2) soil drought experiment
The environmental seed of arabidopsis Col-0 is placed on 4 DEG C of refrigerator vernalization with NaClO sterilization and after 3 days, sows on 1/2MS solid culture medium, grow to choose after 4 days to grow fine and seedling of the same size moves into and fills 8 × 7 native × 6cm
3flowerpot in.The plant (six strains) that each flowerpot is equipped with the soil of identical weight and moves into similar number is to reduce experimental error, all flowerpots are all placed in 22 DEG C of short-day and cultivate, after ten days, stop watering carry out arid process, every other day contain during this time the solution of 0.02%tween-20 and 50 μ M (+)-ABA/AM1/AM2 or 0.02%tween-20 and 0.05%DMSO (contrast) to foliage spray, convert flowerpot position to reduce experimental error simultaneously.After about 12-14 days, recover to water.Equal Taking Pictures recording before and after rehydration.The soil drought experiment of tobacco is similar with arabidopsis, every basin only comprises a strain plant, all plant are all cultivated 24 DEG C of long-day, after one month, stop watering carry out arid process, the solution that every other day contains during this time 0.05%tween-20 and 50 μ M (+)-ABA/AM1/AM2 or 0.05%tween-20 and 0.05%DMSO (contrast) to foliage spray converts flowerpot position simultaneously.Within approximately 14 days, recover afterwards to water.
(3) the in vitro dehydration experiment of blade
The environmental plant of arabidopsis Col-0 growing 1 month under 22 DEG C of short-day conditions is the solution containing 0.02%tween-20 and 50 μ M (+)-ABA/AM1/AM2 or 0.02%tween-20 and 0.05%DMSO (contrast) to foliage spray, continues to place 3 hours.The lotus throne leaf of cutting formed objects is exposed in air after weighing, and timing weighs fresh weight.
(4) cold experiment
The process of seed vernalization and arabidopsis growth is identical with soil drought experiment, three weeks large plants move in 4 DEG C of growth casees places 5 days, and every day is the solution containing 0.02%tween-20 and 50 μ M (+)-ABA/AM1/AM2 or 0.02%tween-20 and 0.05%DMSO (contrast) to foliage-spray.After five days, shifting out incubator is positioned in normal habitat.
II. embodiment
Embodiment 1, micromolecular compound AM1
1, structure
The contained micromolecular compound AM1 of the present invention (ABA mimic1) has the chemical constitution different from ABA, is made up of with a quinoline being connected with amino the dimethylbenzene being connected with sulfonyl, does not have optical activity, as Fig. 1 a.
2, external biochemical test, PP2C phosphoprotein phosphatase activity experiment
External biochemical test shows, AM1 is as a kind of PYL receptor stimulating agent of broad-spectrum high efficacy, can with multiple PYL receptors bind, promote the latter in conjunction with also suppressing PP2C protein phosphatase enzymic activity.
PP2C phosphoprotein phosphatase activity experiment taking SnRK2.6 MALDI-PSD as substrate shows, under the condition that AM1 exists, multiple PYL acceptors can with PP2C phosphoprotein phosphatase (HAB1) combination, thereby (Fig. 1 b), the above results shows that AM1 is a kind of and the similar wide spectrum PYL of ABA receptor stimulating agent for the dephosphorylation of SnRK2.6 MALDI-PSD to suppress HAB1.
3、AlphaScreen
AlphaScreen result shows, AM1 possesses and the similar PYL receptor affinity of ABA and binding ability (Fig. 1 c-e).The AlphaScreen technology for detection AM1 of binding ability by to(for) PYL acceptor and PP2C phosphoprotein phosphatase (HAB1).EC
50value (half effective concentration, the concentration while producing 50% ceiling effect.EC
50be worth lower, the compatibility of compound and acceptor is higher) and signal peak (reading when representation compound and acceptor generation maximum combined, signal peak is higher, the binding ability of compound and acceptor is stronger) show under the condition of AM1 existence, three PYL acceptors, (Fig. 1 c) to comprise PYR1, PYL1 (Fig. 1 d) and PYL2 (Fig. 1 e), all have with ABA and have lower similar compatibility and binding ability with HAB1, significantly be better than Pyrabactin, and three PYL acceptor (PYR1, PYL1 and PYL2) with the dose effect of the binding ability Existence dependency AM1 of HAB1.The above results proves, AM1 is a kind of and the similarly efficient PYL receptor stimulating agent of ABA.
AM1 can promote PYR1 (c), the combination of three PYL acceptors such as PYL1 (d) and PYL2 (e) and phosphoprotein phosphatase HAB1, and there is dose-dependent effect in this interaction.AM1 is all better than Pyrabactin for compatibility (EC50 value) and the bond strength (Photon counts peak value) of three PYL acceptors.
4, yeast two-hybrid experiment
The inventor has carried out yeast two-hybrid experiment, with in conjunction with territory (binding-domain, BD) merge PYL receptor protein and jointly proceed to yeast with the PP2C protein phosphatase zymoprotein of activation domain (activation-domain, AD) combination according to corresponding combination.
Result, as Fig. 2 a, yeast equal energy normal growth on the medium that does not contain isoleucine and tryptophan (Leu/-Trp), shows that yeast conversion is successful.If Fig. 2 b, yeast are not containing isoleucine, in the DMSO control medium of tryptophan and histidine (Leu/-Trp/-His), to grow, the cotransformation bacterial strain of five kinds of PYL acceptors such as PYR1 and PYL1-4 and PP2C phosphoprotein phosphatase cannot normal growth.As Fig. 2 c, yeast are containing 2 μ M ABA and are not containing isoleucine, on the medium of tryptophan and histidine (Leu/-Trp/-His), grow, the cotransformation bacterial strain of five kinds of PYL acceptors such as PYR1 and PYL1-4 and PP2C phosphoprotein phosphatase can normal growth.Show that the interaction of these PYL acceptors and PP2C phosphoprotein phosphatase relies on the existence of ABA.As Fig. 2 d, yeast are containing 2 μ M AM1 and are not containing isoleucine, on the medium of tryptophan and histidine (Leu/-Trp/-His), grow, the cotransformation bacterial strain of four kinds of PYL acceptors such as PYR1 and PYL1-3 and PP2C phosphoprotein phosphatase can normal growth, shows PYL acceptor that AM1 under low concentration can promote that these ABA rely on and the interaction of PP2C phosphoprotein phosphatase.
Result shows, the AM1 of low concentration (2 μ M) can promote the interaction of most PYL acceptors and multiple PP2C phosphoprotein phosphatases, this concentration be only the Pyrabactin of bibliographical information working concentration 1/5.Be different from ABA, the AM1 under this concentration cannot promote the interaction of PYL4 and PP2C phosphoprotein phosphatase, and this point is consistent with protein phosphatase enzymic activity test experience result.The above results further proves, AM1 is a kind of wide spectrum and PYL receptor stimulating agent efficiently.
The structure that embodiment 2, micromolecular compound AM1 and PYL receptor protein and HAB form
The inventor has detected the crystal structure of PYL2-AM1-HAB1 compound, from the three-dimensional structure schematic diagram of compound, oxygen atom on the quinoline of AM1 can be by free hydrone tryptophan (W385) the formation hydrogen bond with HAB1385 position, thereby (Fig. 3 a) for further further HAB1 and PYL2; From the two-dimensional structure partial schematic diagram of compound, AM1 is present in the bag structure of PYL2, is connected that (Fig. 3 b) by many avtive spots of the oxygen atom on sulfonyl and PYL2 with hydrogen bond.Therefore, AM1 is combined in the bag structure of PYL2, by the mechanism similar to ABA, is combined with the key amino acid avtive spot of PYL2 and HAB1 by hydrogen bond, thereby further promotes and the combination of stable PYL2 and HAB1.This result has explained why AM1 is the PYL receptor stimulating agent of broad-spectrum high efficacy.
Embodiment 3, AM1 can cause the gene expression similar to ABA
The inventor has analyzed and has added the impact of AM1 for gene expression in plants.Gene expression analysis shows, adds AM1 and can cause the gene expression similar to ABA (Fig. 4).After 50 μ M AM1 process; in 10 the largest wild type arabidopsis (Col-0) plant in seedling stage, 6 known expressions with environment-stress related gene that are subject to abscisic acid induction significantly increase; approach 50 μ M ABA expression after treatment, and at PYL acceptor three mutant strains (pyr1; Pyl1; Pyl4) in, the expression of corresponding gene increases limitedly, and this result shows that AM1 is undertaken by the receptor-mediated ABA signal path of PYL for the induction of environment-stress related gene that (Fig. 4 a).
RNA order-checking (RNA-seq) result shows, with respect to DMSO control group, respectively have an appointment after the processing expression generation significance of 4500 genes of 50 μ M AM1 and ABA changes, wherein have again exceed 80% gene (3644) be subject to simultaneously AM1 and ABA induction (Fig. 4 b), and be and plant opposing arid, cold that (Fig. 4 c) for the osmotic pressure gene relevant with salt stress the most significantly with respect to control group difference in these genes.This result shows that AM1 can cause the physiological change highly similar to ABA, and mainly concentrates on environment-stress response.
Embodiment 4, AM1 can cause the similar physiological reaction with ABA
(1) arabidopsis drought resistance (or claiming drought resistance)
Physiological Experiment shows, AM1 processing can cause the similar physiological reaction with ABA.In medium, external source is added 1 μ M AM1 and can significantly be suppressed wild type arabidopsis plant (Col-0) seed germination, but PYL acceptor three mutant strain (pyr1; Pyl1; Pyl4) unaffected, this result shows that AM1 is undertaken by the receptor-mediated ABA signal path of PYL for the inhibitory action of seed germination, as Fig. 5.
In order further to explore the impact of AM1 for plant drought resistance, the wild type arabidopsis plant (Col-0) growing in soil two weeks stops feedwater, the plant of choosing formed objects carries out soil drought experiment, every other day spray during this time the aqueous solution that once contains 50 μ M AM1 or ABA, in solution, added the surfactant Tween-20 of 0.02% (v/v) to strengthen the penetration of spray for blade epidermis simultaneously.Through the arid processing of two weeks, spray all survivals after the plant recovery feedwater of ABA, it is 95% withered that the wild type plant that sprays DMSO contrast solution exceedes, and recovers can not restore after feedwater, and the plant that sprays AM1 has 80% still can continued growth, as Fig. 6 a-b.
The corresponding solution of arabidopsis thaliana blade spraying that surrounding is large takes off the blade of formed objects, periodic logging fresh weight after 3 hours.As Fig. 6 c, the rate-of-loss of coolant of excised leaf shows that it is because blade transpiration weakens that the drought resistance of AM1 improves, due to moisture capacity strengthens.This result shows that plant can weaken by spraying AM1 the transpiration of blade in the time running into drought environment, thereby strengthens the drought resistance of plant.
(2) tobacco drought resistance (or claiming drought resistance)
The tobacco (Ben Shi cigarette) growing 1 month under long-day environment stops feedwater, the plant of choosing formed objects carries out soil drought experiment, every other day spray during this time the aqueous solution that once contains 50 μ M AM1 or ABA, in solution, added the surfactant Tween-20 of 0.05% (v/v) to strengthen the penetration of spray for blade epidermis simultaneously.Through the arid processing of two weeks, spray all survivals after the plant recovery feedwater of ABA, the wild type plant major part that sprays DMSO contrast solution is withered, recovers can not restore after feedwater, still can continued growth and spray the plant major part of AM1.
(3) arabidopsis cold resistance (or claiming cold resistance)
In order further to explore the impact of AM1 for plant cold resistance, the wild type arabidopsis plant (Col-0) of three weeks formed objects of growing in soil moves in 4 DEG C of growth casees places 5 days, spray during this time the aqueous solution that once contains 50 μ M AM1 or ABA every day, in solution, added the surfactant Tween-20 of 0.02% (v/v) to strengthen the penetration of spray for blade epidermis simultaneously.
Through cold processing of 5 days, the plant that sprays ABA all survives, the wild type plant that sprays DMSO contrast solution is most of withered (exceeding 70%), retracts normal habitat and can not restore, still can continued growth and spray the plant major part of AM1.
All documents of mentioning in the present invention are all quoted as a reference in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned instruction content of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (14)
1. a purposes for formula (I) compound or its salt, for strengthening stress resistance of plant or the Agrotechnical formulation for the preparation of enhancing stress resistance of plant;
2. purposes as claimed in claim 1, is characterized in that, described resistance is the abiotic stress resistance that ABA participates in.
3. purposes as claimed in claim 2, is characterized in that, the abiotic stress resistance that described ABA participates in be drought resistance, cold resistance, Salt And Alkali Tolerance, resistance to osmotic pressure or stable on heating at least one.
4. purposes as claimed in claim 1, is characterized in that, described plant is the plant that contains the ABA of PYR/PYL family acceptor.
5. purposes as claimed in claim 4, is characterized in that, described plant includes but not limited to: crucifer, plant of Solanaceae, gramineous plants.
6. purposes as claimed in claim 1, it is characterized in that, described plant includes but not limited to: arabidopsis, tobacco, Chinese cabbage, rape, tomato, capsicum, wheat, paddy rice, barley, corn, Chinese sorghum, oat, rye, sugarcane, cotton, soybean, beet, sunflower.
7. a method that strengthens stress resistance of plant, is characterized in that, uses formula (I) compound or its salt to plant:
8. method as claimed in claim 7, is characterized in that, described resistance is the abiotic stress resistance that ABA participates in.
9. method as claimed in claim 8, is characterized in that, the abiotic stress resistance that described ABA participates in be drought resistance, cold resistance, Salt And Alkali Tolerance, resistance to osmotic pressure or stable on heating at least one.
10. method as claimed in claim 7, is characterized in that, described plant is the plant that contains the ABA of PYR/PYL family acceptor.
11. method as claimed in claim 10, is characterized in that, described plant includes but not limited to: crucifer, plant of Solanaceae, gramineous plants.
12. purposes as claimed in claim 7, it is characterized in that, described plant includes but not limited to: arabidopsis, tobacco, Chinese cabbage, rape, tomato, capsicum, wheat, paddy rice, barley, corn, Chinese sorghum, oat, rye, sugarcane, cotton, soybean, beet, sunflower.
13. an Agrotechnical formulation, is characterized in that, described Agrotechnical formulation comprises: formula (I) compound or its salt; And the upper acceptable carrier of agricultural;
14. Agrotechnical formulations as claimed in claim 13, is characterized in that,
The content of formula (I) compound or its salt in Agrotechnical formulation is 1-500 μ M; Be preferably 5-200 μ M; Be more preferably 20-100 μ M; And/or
In described agricultural, acceptable carrier comprises: surfactant.
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| CN106478499A (en) * | 2015-08-28 | 2017-03-08 | 中国科学院上海生命科学研究院 | Strengthen the micromolecular compound of stress resistance of plant |
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| WO2019063009A1 (en) * | 2017-09-30 | 2019-04-04 | 中国科学院上海生命科学研究院 | Method for improving rice yield by jointly knocking out aba receptor pyl family genes and use thereof |
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| WO2019025153A1 (en) | 2017-07-31 | 2019-02-07 | Bayer Cropscience Aktiengesellschaft | Use of substituted n-sulfonyl-n'-aryl diaminoalkanes and n-sulfonyl-n'-heteroaryl diaminoalkanes or salts thereof for increasing the stress tolerance in plants |
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| CN113207886A (en) * | 2021-04-15 | 2021-08-06 | 湖南农业大学 | Application of benconazole serving as strigolactone inhibitor |
| CN113475506A (en) * | 2021-05-21 | 2021-10-08 | 广东海洋大学 | Composition for reducing sugarcane transpiration and improving drought resistance and application technology thereof |
| WO2022268520A1 (en) | 2021-06-21 | 2022-12-29 | Bayer Aktiengesellschaft | Use of substituted pyrrolidinones or their salts for increasing stress tolerance of plants |
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