CN102533846A - Method for increasing the resistance of plants to hypoxic conditions - Google Patents
Method for increasing the resistance of plants to hypoxic conditions Download PDFInfo
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- CN102533846A CN102533846A CN2012100036343A CN201210003634A CN102533846A CN 102533846 A CN102533846 A CN 102533846A CN 2012100036343 A CN2012100036343 A CN 2012100036343A CN 201210003634 A CN201210003634 A CN 201210003634A CN 102533846 A CN102533846 A CN 102533846A
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Abstract
Methods are provided for increasing the resistance of plants to hypoxic or anoxic conditions. Such methods may be applied to increase the penetrance of plant roots in the growth medium or into soil. The methods according to the invention may include providing plants with a stress tolerance gene. Similar effects can be obtained by applying chemical compounds, including neonicotinoid compounds, to the plants.
Description
The application one Chinese patent application 200680021382.X that is entitled as " improving the method for plant to the hypoxia condition resistance " that to be the contriver submit on June 6th, 2006 divides an application.
The present invention is provided for improving the method for plant to hypoxemia or oxygen free condition resistance.These class methods can be used for promoting roots of plants to penetrate growth medium or soil.Method of the present invention can comprise the modification to Plant Genome, and said modification is through providing external source stress tolerance gene or corresponding to the stress tolerance variant of the native gene of this type foreign gene to plant.Method of the present invention also can comprise plant or its habitat; Or to its cell or seed use anabasine compound (neonicotinoid compound), such as but not limited to Provado (imidacloprid), Ti304 (nitenpyram), acetamiprid (acetamiprid), thiophene worm quinoline (thiacloprid), thiophene worm piperazine (thiamethoxam), clothianadin (clothianidin) and MTI-446 (dinotefuran).Especially effectively anabasine compound is the anabasine compound that comprises chloropyridine (chloropyridine) side chain; For example Provado, Ti304, acetamiprid and thiophene worm quinoline; Particularly its degraded in plant can discharge 6-chlorine apellagrin (6-chloronicotininc acid; Those anabasine compounds 6-CNA) are as for example Provado and thiophene worm quinoline.Plant or its habitat also can directly be handled with 6-CNA.
Background technology
The plant that transform stress-tolerance as is known in the art.The stress tolerance of vegetable cell and plant can be for example through reduce endogenous gathering-ADP-ribose polymerase (PARP) or the activity or the level of gathering (ADP-ribose) glycogen lytic enzyme (PARG) realize, it is described in respectively among WO 00/04173 and the WO04/090140.
European patent application No.04077624.7 has described and has used following nucleotide sequence for example in plant, to cross the stress tolerance that expression realizes plant and vegetable cell, and said nucleotide coding is participated in the enzyme of NAD salvage route and/or NAD de novo synthesis.
Yet the stress tolerance gene that these files all do not have public use wherein to mention obtains the possibility to the tolerance of hypoxemia or oxygen free condition in vegetable cell and plant.They do not disclose the purposes that wherein said stress tolerance gene is used for following purpose yet: allow the root system of plant deeper to penetrate into growth medium or soil.
Except that insect control, the application of anabasine compound other purposes in plant also is (WO 01/26468, and WO 03/096811) known in the art.
WO 01/26468 discloses the method for improving plant-growth, and it comprises to plant or its location uses at least a compound that is selected from anabasine.
Output of the agronomy plant that WO03/096811 has described in following place and/or vigor can improve through the seed of handling plant with anabasine compound or improve, and the insect pest level is lower than the indicated use of insecticide that needs and is used for the level that insect is controlled purpose in the said place.Said method is considered to be applicable to non-transgenic plant and the plant with foreign gene that said foreign gene coding is through the generation of Bacillus thuringiensis (Bacillus thuringiensis) the delta-endotoxin proteins matter of modification.
Yet these files are not all described in order to improve vegetable cell or the plant tolerance to hypoxemia or oxygen free condition, or allow the root system of plant deeper to penetrate into the purpose of growth medium or soil, anabasine compound are used for the purposes of plant.
Therefore; This area does not relate to the penetration depth that increases root system of plant or root entering growth medium or soil yet; Or improve vegetable cell or plant method to the tolerance of hypoxemia or anaerobic stress conditions; Said method such as hereinafter are using the stress tolerance gene described in different embodiments and the claim, or through plant or its cell are used anabasine compound.
Summary of the invention
One embodiment of the invention provide and improve vegetable cell or the plant novel method to hypoxemia or oxygen free condition tolerance; Said method comprises to vegetable cell or plant provides stress tolerance to strengthen transgenic, and wherein said stress tolerance strengthens transgenic and is selected from:
-can reduce the stress tolerance enhancing transgenic of plant endogenous PARP genetic expression, particularly wherein said transgenes encoding PARP inhibitory RNA molecules;
-can reduce the stress tolerance enhancing transgenic of plant endogenous PARG genetic expression, particularly wherein said transgenes encoding PARG inhibitory RNA molecules; Or
The stress tolerance of the plant function enzyme of-coding Reduced nicotinamide-adenine dinucleotide salvage route strengthens transgenic, and said enzyme is selected from nicotinamidase, nicotinate/ester phosphoribosyl transferase (nicotinate phosphoribosyltransferase), NAMN adeninyl transferring enzyme or Reduced nicotinamide-adenine dinucleotide synthetic enzyme.
In another embodiment, the present invention relates to this type stress tolerance enhancing transgenic and promote that roots of plants penetrates the growth medium purposes of (comprising soil).
Another embodiment of the present invention is provided for improving vegetable cell or the plant method to hypoxemia or oxygen free condition tolerance, and it comprises to vegetable cell, plant, maybe will grow up to seed of this type of plant or the anabasine compound that significant quantity is used in its habitat.
In another embodiment, the present invention relates to this compounds and promote that roots of plants penetrates the growth medium purposes of (comprising soil).
The invention still further relates to and be used to improve vegetable cell or plant, comprise the step that the 6-chlorine apellagrin of significant quantity is offered the cell of said plant the method for hypoxemia or oxygen free condition tolerance.
The invention still further relates to 6-CNA and be used for promoting that roots of plants penetrates into the purposes of growth medium.
Description of drawings
Fig. 1: the synoptic diagram of measuring the dark assay method of the roots of plants grow in the agar-agar soln.Fill with transparent or translucent growth medium (3) and to have the container (1) that seals (2), said growth medium is for example agar-water of 0.4% or agar-water of 0.7%, wherein can add extra test compounds.Xiang Guanzhong adds a seed of sprouting in advance, and allows it to grow for three weeks.After three weeks were grown in the vertical position, the valley from the substratum top to root was measured the root dark (5) of plant (4).
Fig. 2: the case shape diagram of Arabidopis thaliana (Arabidopsis thaliana) cv.Col-0 roots of plants dark (mm); Said plant is compared with non-transgenic arabidopsis thaliana (Col-0) and comprises transgenic, and said transgenes encoding can reduce the dsRNA molecule of endogenous PARP2 genetic expression.
Analyze following population:
Col-0: data indication wild-type Arabidopis thaliana strain system
427-16: the data indication has the Arabidopis thaliana transgenic line of comprising of weak tolerance of anti-PARP2 gene to the high light stress conditions
427-20: the data indication has the Arabidopis thaliana transgenic line of comprising of weak tolerance of anti-PARP2 gene to the high light stress conditions
427-20: the data indication has the Arabidopis thaliana transgenic line of comprising of medium tolerance of anti-PARP2 gene to the high light stress conditions
The representative case shape figure (or case shape figure and palpus box figure) of each class value has been represented in this figure left side, the statistical measure below said value has been summed up:
-median
-last quartile and lower quartile
-minimum and maximum data value.
In addition, on the right of each group numerical value, indicate the average and the average value standard deviation of these numerical value.
Case shape figure explains as follows:
50% data in the middle of-case self comprises.The upper limb of case (joint) is pointed out the 75th percentile of DS, and following joint is pointed out the 25th percentile.The scope of middle two quartiles is known as interquartile range.
The median of the line designation data the in-case.
-must the minimum and maximum data value of end indication, exist except the situation of outlier (outlier), must extend to value point nearest in the scope of 1.5 times of interquartile ranges in this case.
Fig. 3: the case shape synoptic diagram and the average value standard deviation of dark (mm) observed value of Arabidopis thaliana cv.Col-0 roots of plants, said plant comprises transgenic, and said transgenes encoding can reduce the dsRNA molecule of endogenous PARP genetic expression.
Analyze following population:
Col-0: data indication wild-type Arabidopis thaliana strain system
427-22: the data indication has the Arabidopis thaliana transgenic line of comprising of height endurability of anti-PARP2 gene to the high light stress conditions
427-24: the data indication has the Arabidopis thaliana transgenic line of comprising of low tolerance of anti-PARP2 gene to the high light stress conditions
Fig. 4: the case shape synoptic diagram and the average value standard deviation of dark (mm) observed value of Arabidopis thaliana cv.C24 roots of plants, said plant comprises transgenic, and said transgenes encoding can reduce the dsRNA molecule of endogenous PARP genetic expression.
Analyzed following population:
C24: data indication wild-type Arabidopis thaliana strain system
1599: the data indication has the Arabidopis thaliana transgenic line of comprising of height endurability of anti-PARP2 gene to the high light stress conditions
1463: the data indication has the Arabidopis thaliana transgenic line of comprising of medium tolerance of anti-PARP2 gene to the high light stress conditions
1681: the data indication has the Arabidopis thaliana transgenic line of comprising of medium tolerance of anti-PARP1 gene to the high light stress conditions
1690: the data indication has the Arabidopis thaliana transgenic line of comprising of medium tolerance of anti-PARP1 gene to the high light stress conditions
Fig. 5: case shape synoptic diagram and the average value standard deviation of treating dark (mm) observed value of root that separates the measurement of the genetically modified Arabidopis thaliana Col-0 of anti-PARP2 population.
Population below having analyzed:
Azygous (Azygous): the data indication is from the arabidopsis thaliana of the population that does not contain anti-PARP2 gene
Genetically modified: the data indication is from the arabidopsis thaliana of the population that contains anti-PARP2 gene
Fig. 6: the Arabidopis thaliana cv.C24 plant with the Provado of multiple concentration is handled is compared with untreated Arabidopis thaliana cv.C24 plant, the case shape synoptic diagram and the average value standard deviation of dark (mm) observed value of root.
Population below having analyzed:
0: untreated Arabidopis thaliana C24 plant
50: with the Arabidopis thaliana C24 plant of 50 mg/litre Provados processing
100: with the Arabidopis thaliana C24 plant of 100 mg/litre Provados processing
Fig. 7: the Arabidopis thaliana cv.C24 plant with the 6-chlorine apellagrin of multiple concentration is handled is compared with untreated Arabidopis thaliana cv.C24 plant, the case shape synoptic diagram and the average value standard deviation of dark (mm) observed value of root.
Population below having analyzed:
0: untreated Arabidopis thaliana C24 plant
1: with the Arabidopis thaliana C24 plant of 1 mg/litre 6-chlorine apellagrin processing
5: with the Arabidopis thaliana C24 plant of 5 mg/litre 6-chlorine apellagrins processing
Detailed Description Of The Invention
The present invention is based on following understanding: comprise the stress tolerance gene (mosaic gene of the following dsRNA that for example encodes; Said dsRNA purpose is to make plant parp1 or the parp2 expression of gene reticent) plant produced following root system, the root of said root system compare with the root of control plant stretch in the growth medium darker.Of prior art, the roots of plants that comprises the stress tolerance gene penetrates darker phenomenon not to be noted yet, and needs exploitation concrete assay method as described herein to be used for the penetrance that the statistical analysis root gets into substratum.
Although be not intended to the present invention is not limited to the concrete mode of action; Think that still the stress tolerance gene improves the tolerance (comprising the tolerance of the vegetable cell of root to hypoxemia and oxygen free condition) of vegetable cell; Thereby the root that allows to comprise this type stress tolerance gene is grown under not good oxygen condition; As can in the darker regional or darker pedosphere of growth medium, find that the there oxygen tension is lower.The penetrance that root system of plant gets into darker edaphic raising has partly been explained in the open air under the condition containing the arid resistance that the viewed plant of stress tolerance gene described herein (for example following dsRNA, said dsRNA coding makes endogenous parp1 or the reticent gene of parp2 genetic expression) is improved.
After adding anabasine compound or 6-chlorine apellagrin, can in said assay method, observe the similar effect of root elongation.The use of anabasine has nothing to do to the effect of the root growth degree of depth and the existence of insect, and said insect is the target of above-mentioned anabasine.Therefore, this effect is also with plant or vegetable cell or to grow up to the biological chemistry improvement of stress tolerance (particularly with hypoxemia or the relevant stress tolerance of anaerobic) of the seed of plant relevant.
Therefore, in first embodiment, the present invention points to stress tolerance and strengthens transgenic raising vegetable cell, plant or the seed purposes to the tolerance of hypoxemia or oxygen free condition.
" hypoxemia or the oxygen free condition " that this paper uses is meant the condition that part exposed of vegetable cell, plant or this type plant, and wherein the operability of oxygen is low or very low.Oxygen free condition is meant the condition that almost can not obtain oxygen.Usually, the condition that dissolved oxygen concentration is lower than about 2 mg/litre is called as hypoxemia (0.1 mg/litre is to 2 mg/litre), and dissolved oxygen is lower than 0.1 mg/litre, and the condition that particularly is lower than 0.05 mg/litre is called as anaerobic.Normal dissolved oxygen concn is about 8 mg/litre in the water.Hypoxia condition in the soil is meant that oxygen is brought down below 5% condition in the condition that oxygen tension is low, particularly soil atmosphere.
When hypoxia condition can betide plant or plant part and is submerged.Hypoxia condition also can betide oxygen consumption when high, for example in the pedosphere of the organic debris in comprising a large amount of microbial metabolisms.In addition, hypoxia condition betide growth medium than deep layer, wherein oxygen spreads from the surface.Hypoxia condition also betides soil than deep layer, because the oxygen tension specific surface reduction that oxygen spreads and causes.The speed that oxygen reduces depends on the existence, water-content of the tightness (compactness) (soil is tight more, and the soil atmosphere of existence is few more) of soil, the organic materials in decomposing etc.
" stress tolerance enhancing transgenic " that this paper uses is meant such transgenic; Promptly working as said transgenic is introduced in vegetable cell or the plant; Or when in vegetable cell or plant, expressing; Better stress tolerance is provided for cell or plant; Said stress tolerance is for example through using compound (weedicide, mycocide, sterilant, plant-growth regulator, adjuvant, fertilizer), be exposed to abiotic stress (for example arid, waterlogging, flood, high light condition, strong UV radiation, the hydrogen peroxide level of raising, extreme (high or low) temperature, ozone and other atmospheric polluting materials, soil salinity or heavy metal, hypoxemia, anaerobic or the like) or biology to coerce (for example pathogenic agent or insect infection comprises fungi infestation, virus infection, infectation of bacteria, insect infection, nematode infections, mycoplasma infection and mycoplasma appearance biological infection etc.) and be endowed plant.
This type stress tolerance strengthens transgenic can reduce poly in vegetable cell or the plant (ADP-ribose) polysaccharase (PARP) expression of gene and/or active transgenic described in WO 00/04173 or EP04077984.5 (quoting as a reference at this paper).
Poly (ADP-ribose) polysaccharase (PARP) is also referred to as poly (ADP-ribose) transferring enzyme (ADPRT) (EC 2.4.2.30); Be the ribozyme that is found in most eukaryotes, said eukaryote comprises vertebrates, arthropods, mollusk, sline cungi (slime mould), ditch volume algae (dinoflagellates), fungi and other lower eukaryotes except that yeast.Said enzymic activity has also obtained proof (people such as Payne, 1976 in a large amount of plants; Willmitzer and Wagner, 1982; People such as Chen, 1994; O ' Farrell, 1995)
The main catalysis of PARP is from NAD
+The conversion of the ADP-ribose part carboxyl of glutaminic acid residue in target protein, and ADP-ribose polymerization subsequently.Main target protein is PARP self, but histone, high mobility chromosomoid protein (high mobility groupchromosomal protein), topoisomerase, endonuclease and archaeal dna polymerase also are shown as and can receive this modification.
As concrete embodiment, stress tolerance strengthens the dna fragmentation that transgenic can comprise following effective connection:
A) the effable promotor of plant;
B) obtained the DNA district of following RNA molecule after quilt is transcribed, said RNA molecule (PARP inhibitory RNA molecules) can reduce the expression of the endogenous PARP encoding sox of plant;
C) the DNA district of participation Transcription Termination and polyadenylic acidization.
After transcribing in the district, said DNA can produce so-called antisense rna molecule; Said antisense rna molecule is with the mode of transcribing or transcribe the expression that the back mode reduces PARP encoding sox in target plant or the target vegetable cell; Said antisense rna molecule comprises at least 20 or 21 successive Nucleotide, and the complementary sequence of the PARP encoding sox nucleotide sequence that exists in said continuous nucleotide and said vegetable cell or the plant has at least 95% to 100% sequence identity.
Said DNA district also can produce the so-called adopted RNA molecule that has; Said have adopted RNA molecule with the mode of transcribing or transcribe the back mode and reduce the expression of PARP encoding sox in target plant or the target vegetable cell; Said have adopted RNA molecule to comprise at least 20 or 21 successive Nucleotide, and the nucleotide sequence of the PARP encoding sox that exists in said continuous nucleotide and said vegetable cell or the plant has at least 95% to 100% sequence identity.
Yet, the antisense of the PARP coding region of about 20nt or have the minimum nucleotide sequence in adopted RNA district can be included in the bigger RNA molecule, said bigger RNA bulk of molecule 20nt to and target gene size equal lengths between change.Therefore, said antisense or adopted Nucleotide district is arranged can be about 21nt grows to about 5000nt, for example length is 21nt, 40nt, 50nt, 100nt, 200nt, 300nt, 500nt, 1000nt, 2000nt or even about 5000nt or longer.In addition, the object of the invention does not require that nucleotide sequence or the genetically modified coding region of employed inhibition PARP RNA molecule and endogenous PARP gene are identical or complementary, said endogenous PARP expression of gene by target in vegetable cell, to reduce.Sequence is long more, and is more not strict to the requirement of whole sequence identity.Therefore, there are justice or antisense district can have the whole sequence identity with the nucleotide sequence of endogenous PARP gene or its complementary sequence about 40% or 50% or 60% or 70% or 80% or 90% or 100%.Yet of preamble, antisense or have adopted district should comprise the nucleotide sequence of 20 continuous nucleotides, the nucleotide sequence of itself and endogenous PARP gene has about 100% sequence identity.Preferably, one of about 100% sequence identity section sequence should be about 50,75 or 100nt.
With regard to the object of the invention, " the sequence identity " of two related nucleotide sequences representing with per-cent is meant that the positional number that has identical residue in the sequences of two best comparisons is divided by the positional number that is compared (* 100%).The room is considered to have the position of residue inequality, and promptly there is residue in sequence and does not have the position in the comparison of this residue in another sequence.The comparison of two sequences is carried out through Needleman and Wunsch algorithm (Needleman and Wunsch 1970) area of computer aided sequence alignment; (it is Wisconsin Package 10.1 versions (Genetics Computer Group can to use standard software program such as GAP; Madison; Wisconsin, U.S.) a part), use acquiescence rating matrix (room produces point penalty 50 and extends point penalty 3 with the room) to come to carry out expediently.
Be understood that when in a single day the nucleotide sequence of the corresponding dna molecular of reference defined the nucleotide sequence of RNA molecule, the thymus pyrimidine in the nucleotide sequence (T) should use uridylic (U) to replace.The application's context can clearly be that reference is with RNA or DNA.
The effectiveness that above-mentioned transgenic reduces endogenous PARP genetic expression can be through comprising the DNA element be further strengthened, unusual, the not expression of the PARP inhibitory RNA molecules of polyadenylic acidization that said DNA element causes.Be applicable to the DNA district of this type DNA element of this purpose for the coding self-splicing ribozyme described in WO00/01133A1.
Described in WO 99/53050A1; The effectiveness that above-mentioned transgenic reduces the endogenous PARP genetic expression of vegetable cell also can further strengthen through following mode: in a vegetable cell, comprise the transgenic of encoding antisense PARP inhibitory RNA molecules described herein and the transgenic that coding described herein has adopted PARP inhibitory RNA molecules simultaneously, wherein said antisense and have the adopted PARP inhibitory RNA molecules can be through the formation of the base pairing between said at least 20 continuous nucleotides double-stranded RNA district.
Like what in WO 99/53050A1, further described, the have justice and the antisense PARP inhibitory RNA district that can form the double-stranded RNA district may reside in the RNA molecule, preferably are spaced apart to distinguish and leave.Transcribed spacer can comprise intron sequences.This type transgenic can be described below expediently and to make up: be connected effectively comprising in the inverted repeats to distinguish with 3 ' the end formation of participating in Transcription Termination and polyadenylic acidization with the effable promotor of plant from the dna fragmentation (it is expressed by target and reduces) of at least 20 Nucleotide of isolating or the endogenous PARP gene identified.In order to realize the genetically modified structure of this type, can use the carrier of describing among the WO 02/059294A1.
Existing nomenclature is called PARP1 protein (with corresponding parp1 gene) with the polysaccharase that zinc refers to that contains of classics; And non-classical PARP protein is called as PARP2 (with corresponding parp2 gene) now on the structure, and " PARP encoding sox " that this paper uses can refer to the arbitrary type in the two.
The following experimental evaluation database login number (quoting as a reference at this paper) with poly polymerase protein sequence, its part or homologous sequence supposition that confirm can be used according to the invention: BAD53855 (rice (Oryza sativa)); BAD52929 (rice); XP_477671 (rice); BAC84104 (rice); AAT25850 (corn (Zea mays)); AAT25849 (corn); NP_197639 (Arabidopis thaliana); NP_850165 (Arabidopis thaliana); NP_188107 (Arabidopis thaliana); NP_850586 (Arabidopis thaliana); BAB09119 (Arabidopis thaliana); AAD20677 (Arabidopis thaliana); Q11207 (Arabidopis thaliana); C84719 (Arabidopis thaliana); T51353 (Arabidopis thaliana); T01311 (Arabidopis thaliana); AAN12901 (Arabidopis thaliana); AAM13882 (Arabidopis thaliana); CAB80732 (Arabidopis thaliana); CAA10482 (Arabidopis thaliana); AAC79704 (corn): AAC19283 (Arabidopis thaliana); CAA10888 (corn); CAA10889 (corn); CAA88288 (Arabidopis thaliana).
As the concrete embodiment of the present invention, the gene that reduces PARP genetic expression can comprise the dna fragmentation of following effective connection:
A) the effable promotor of plant;
B) by the DNA district of generation RNA molecule after being transcribed, said RNA molecule comprises:
A. antisense base sequences; It comprises at least about 20 successive Nucleotide; Said successive Nucleotide has about 96% sequence identity: SEQ ID 1 (Arabidopis thaliana parp1 coding region), SEQ ID 2 (Arabidopis thaliana parp 2 coding regions), SEQ ID 3 (corn parp1 coding region), SEQ ID 4 (another corn parp1 coding region), SEQ ID 5 (corn parp2 coding region) or the nucleotide sequence of SEQ ID 6 (cotton parp2 Partial cDNA) or the nucleotide sequence of coded protein with the nucleotide sequence that is selected from following about 20 continuous nucleotides, and said protein has and the said nucleotide sequence coded similar or identical aminoacid sequence of aminoacid sequence.
B., sense nucleotide sequence is arranged, and it comprises at least about 20 and antisense base sequences complementary Nucleotide.Therefore; There is sense nucleotide sequence can comprise sequence at least about 20 continuous nucleotides; Said sequence has about 96% sequence identity: SEQ ID 1 (Arabidopis thaliana parp1 coding region), SEQ ID 2 (Arabidopis thaliana parp 2 coding regions), SEQ ID 3 (corn parp1 coding region), SEQ ID 4 (another corn parp1 coding region), SEQ ID5 (corn parp2 coding region) or the nucleotide sequence of SEQ ID 6 (cotton parp2 Partial cDNA) or the nucleotide sequence of coded protein with the nucleotide sequence that is selected from following about 20 continuous nucleotides, and said protein has and the said nucleotide sequence coded similar or identical aminoacid sequence of aminoacid sequence;
Wherein there are justice and antisense base sequences can form double stranded rna molecule (dsRNA);
C) be used for the DNA district of Transcription Termination and polyadenylic acidization.
Yet, be understood that can use and reduce the gene of PARP genetic expression like other that describe among WO 00/04173 or the EP 04077984.5.
In another embodiment of the present invention, it can be that the PARG encoding sox that can reduce plant or vegetable cell is expressed and/or active transgenic that stress tolerance strengthens transgenic, described in WO2004/090140 (quoting as a reference at this paper).
PARG (poly (ADP-ribose) glycogen lytic enzyme; E.C.3.2.1.143) will gather (ADP-ribose) polymkeric substance through its exoglycosidase and ENDOGLYCOSIDASES active (PARG) and be converted into free ADP-ribose.
In plant; Gather (ADP-ribose) glycogen lytic enzyme inactivation in two mutants through based on gene mapping the clone of wild type gene having been identified, said two mutants receives clock control genetic transcription in the Arabidopis thaliana and grows into the influence of the photoperiod dependency conversion (tej) of blooming from vegetalitas.The nucleotide sequence of this gene can be at accession number AF394690 (people such as Panda, 2002 Dev.Cell.3,51-61; SEQ ID No 7) from the Nucleotide DB, obtains under.
Other are found in WO 2004/090140A2 from the nucleotide sequence of the plant PARG encoding sox of plant and from the method that other plant separates other PARG encoding soxs and variant thereof, and this nucleotide sequence is for example from the PARG gene (SEQ ID No8) of yam (Solanum tuberosum); Rice (SEQ ID No 9) or corn (SEQ ID No 10).
Therefore, in one embodiment, transform as the dna fragmentation that anti-plant of coercing or vegetable cell can comprise following effective connection:
A) the effable promotor of plant;
B) when the DNA district that is produced inhibitory RNA molecules after transcribing, said RNA molecule comprises:
I. GEM 132 district; It comprises at least about 20 successive Nucleotide; Said successive Nucleotide has at least 96% sequence identity with the nucleotide sequence that is selected from following about 20 continuous nucleotides: the complementary sequence of the proteic nucleotide sequence of coded plant PARG (the for example nucleotide sequence of SEQ ID 7, SEQ ID 8, SEQ ID 9 or SEQ ID 10); Or the nucleotide sequence of coded protein, said protein has the aminoacid sequence similar or identical with said nucleotide sequence; Or
Ii., adopted Nucleotide district is arranged; It comprises at least about 20 and is selected from following continuous nucleotide: the proteinic nucleotide sequence of coded plant PARG (the for example nucleotide sequence of SEQ ID 7, SEQ ID 8, SEQ ID 9 or SEQ ID 10); Or the nucleotide sequence of coded protein, said protein has the aminoacid sequence similar or identical with said nucleotide sequence; Or
Iii. like i) or ii) described antisense with sense nucleotide sequence arranged, wherein said antisense with have sense nucleotide sequence can form double stranded rna molecule;
C) the DNA district of participation Transcription Termination and polyadenylation.
Those skilled in the art will can understand at once, the length of justice and antisense base sequences or dsRNA molecule is arranged, and other parameters of the sequence identity of ParG inhibitory RNA molecules can be used for the PARP inhibitory RNA molecules as stated.
In another embodiment of the present invention, stress tolerance strengthens the transgenic that transgenic can be the plant function enzyme of coding Reduced nicotinamide-adenine dinucleotide salvage route.Therefore, the stress tolerance enhancing gene can comprise the dna molecular of following effective connection of (quoting as a reference at this paper) described in EP 04077624.7:
A) the effable promotor of plant;
B) the DNA district of the plant function enzyme of coding Reduced nicotinamide-adenine dinucleotide salvage route, said enzyme is selected from nicotinamidase, nicotinate/ester phosphoribosyl transferase, NAMN adeninyl transferring enzyme or Reduced nicotinamide-adenine dinucleotide synthetic enzyme; With
C) 3 ' end region of participation Transcription Termination and polyadenylation.
" the plant function enzyme of Reduced nicotinamide-adenine dinucleotide salvage route " that this paper uses is such enzyme, promptly in being introduced into plant, with can be in vegetable cell after suitable controlling element (for example effable promotor of plant and termination subarea) is connected quilt transcribed and translated and produced the enzyme of the NAD salvage route of function.This enzyme comprise derive from plant origin remedy synthetic enzyme (and encoding sox) from NAD, and derive from yeast (yeast saccharomyces cerevisiae (Saccharomyces cereviseae)) or derive from the enzyme of other yeast or fungi.Think the back protein in addition can more be applicable to method of the present invention because they can not receive enzyme feedback regulation etc., and similarly can receive said adjusting from the enzyme of plant.
The enzyme of participating in the NAD salvage route comprises following:
-nicotinamidase (EC 3.5.1.19), the carboxamido-group hydrolysis of its catalysis vitamin PP, thus discharge nicotinic acid root and NH3.This enzyme also is known as vitamin PP desaminase, vitamin PP Ntn hydrolase, YNDase or vitamin PP hydroamidase;
-nicotinate/ester phosphoribosyl transferase (EC 2.4.2.11), it also is known as nicotinic acid ribo nucleo tidase, NAMN glycogen lytic enzyme; Nicotinate mononucleotide pyrophosphorylase; The nicotinic acid phosphoribosyl transferase; Reaction below its catalysis
Nicotinate/ester-D-ribonucleotide+diphosphate=nicotinate/ester+5-phosphoric acid-α-D ribose 1-diphosphate
-nicotinic acid adenosine nucleotide acyltransferase (EC 2.7.7.18) also is known as acid amides-NAD
+Pyrophosphorylase; The NAMN adenylyl transferase; Remove acid amides Reduced nicotinamide-adenine dinucleotide pyrophosphorylase; NaMT-ATase; The NAMN adenylyl transferase; Reaction below its catalysis
ATP+ nicotinic acid ribonucleotide=diphosphate+go carboxamido-group-NAD
+
-NAD-synthase (EC 6.3.1.5) also is known as the NAD synthetic enzyme; NAD
+Synthase; The Reduced nicotinamide-adenine dinucleotide synthetic enzyme; Diphosphopyridine nucleotide synthetase; Reaction below its catalysis
Remove carboxamido-group-NAD
++ ATP+NH3=AMP+ diphosphate+NAD
+
In one embodiment of the invention; The DNA district of the plant function enzyme of coding NAD salvage route can comprise from SEQ ID No11,12,13,14 or 15 nucleotide sequence or the nucleotide sequence of the following proteins of encoding, and said protein has the protein similar or identical aminoacid sequence coded with above-mentioned nucleotide sequence.
Like Hunt etc., 2004 is said, and the plant homologue of these enzymes has been identified, and these dna sequence dnas can be used to similar effect (people such as Hunt, 2004, New Phytologistl63 (I): 31-44).Dna sequence dna through identifying has following accession number: nicotinamidase is At5g23220 (SEQ ID No16); At5g23230 (SEQ ID No 17) and At3gl6190 (SEQ ID No 18); Nicotinate/ester phosphoribosyl transferase is At4g36940 (SEQ ID No 19), At2g23420 (SEQ ID No20); NAMN adeninyl transferring enzyme is At5g55810 (SEQ ID No 21), and the NAD synthetic enzyme is At1g55090 (SEQ ID No 22).
Yet, be understood that to be transform as the variant that anti-plant of coercing also can comprise these nucleotide sequences that it comprises insertion, disappearance and replaces.Same, can use homologue from the said nucleotide sequence of species except that yeast saccharomyces cerevisiae.These include but are not limited to from the nucleotide sequence of plant and encode has the proteinic nucleotide sequence of same acid sequence, and the variant of this type nucleotide sequence.
The variant of said nucleotide sequence with should have preferably at least about 80% or 85% or 90% or 95% sequence identity through the nucleotide sequence identified (its coding is remedied the enzyme of salvage pathway, the sequence of for example differentiating in the sequence table from NAD).Preferred these variants are encoding function property protein, its have with from the identical enzymic activity of the enzyme of NAD salvage pathway.
After the reading preceding text strengthen transgenic raising vegetable cell, plant or the description of seed to the purposes of the present invention of hypoxemia or oxygen free condition tolerance to stress tolerance; Those skilled in the art can understand at once that the variant that can use corresponding this type stress tolerance to strengthen genetically modified native gene obtains similar effect, and said variant makes the vegetable cell or the plant that have this type variant that higher stress tolerance arranged.For example, the variant of plant endogenous parp2 gene (it has lower expression level, and the stress tolerance of raising is provided for the plant with this variant) can be to use with the similar mode of transgenic that reduces endogenous parp2 genetic expression.This type variant gene can be introduced into vegetable cell or plant through breeding technique.
Those skilled in the art can understand that also different stress tolerance enhancing gene or genetically modified expression can cause a group different events, and this shows that different effects distributes---from almost there not being the effect of effect to highly significant.Yet those skilled in the art obviously can distinguish, differentiate or separate the colony's representative that meets the needs of most.
The present invention of another embodiment is provided for promoting that roots of plants penetrates into the method for growth medium or soil; It comprises provides stress tolerance to strengthen transgenic or these stress tolerances strengthen genetically modified endogenous variant to plant, like this paper at described in its different embodiment those.
" roots of plants elongation " or " roots of plants penetrates into growth medium or soil " that this paper uses is meant the degree of depth (also referring to Fig. 1) that the measured root of valley from the growth media surface to the root is grown in solid growth culture media (comprising soil).
Usually; " roots of plants elongation or the raising that penetrates " is meant that the statistics at least of the root growth degree of depth in the measured growth medium of the valley from the media surface to the root growth improves significantly; It also can be measured as wild-type with reference to the difference in the comparison that roots of plants is dark and the roots of plants that is transform as stress-tolerance is dark, or is measured as with the difference in the comparison that the roots of plants of special compound treatment is dark and the roots of plants that is untreated is dark.
For correct understanding the present invention, understand that following content is important: the root system of plant that reaches through the inventive method deeper penetrates into and is not equal to the raising of root system on volume or dry weight or fresh weight in growth medium or the soil.In fact, the volume of root system can be significantly increased, but root all is in very shallow table place under growth medium or the soil surface.On the contrary, the roots of plants of handling according to the present invention size, volume, weight or even length on can equate, but deeper stretch under the surface of growth medium or soil.
" growth medium " that this paper uses is intended to relate to any substratum that is applicable to plant-growth, comprises soil.This type substratum comprises the liquid of solidified or gelation, for example water-agar, peat (peat), the peat composed of rotten mosses (turf), dissimilar soil etc.
In another embodiment, the present invention points to anabasine compound raising vegetable cell, plant or the seed purposes to hypoxemia or oxygen free condition tolerance.Therefore; The present invention provides and is used for improving vegetable cell, plant or the seed method to hypoxemia or oxygen free condition tolerance, and it comprises the step of the anabasine compound of the formula (I) of using significant quantity to the habitat of vegetable cell, plant, seed or plant or to growth medium.
Wherein
Het representes heterocycle, and said heterocycle is optional by fluorine, chlorine, methyl or ethyl list replacement or polysubstituted under each situation, and said heterocycle is selected from down the group heterocycle:
Pyridin-3-yl, pyridine-5-base, 3-pyridyl (pyridinio), 1-oxidation-5-pyridyl (1-oxido-5-pyridinio), 1-oxidation-5-pyridyl, THF-3-base, thiazole-5-base,
A representes C
1-C
6-alkyl ,-N (R
1) (R
2) or S (R
2),
Wherein
R
1Expression hydrogen, C
1-C
6-alkyl, phenyl-C
1-C
4-alkyl, C
3-C
6-naphthenic base, C
2-C
6-alkenyl or C
2-C
6-alkynyl, and
R
2Expression C
1-C
6-alkyl, C
2-C
6-alkenyl, C
2-C
6-alkynyl ,-C (=O)-CH
3Or benzyl,
R representes hydrogen, C
1-C
6-alkyl, C
2-C
6-alkenyl, C
2-C
6-alkynyl ,-C (=O)-CH
3Or benzyl, or and R
2Represent following group together:
-CH
2-CH
2-,-CH
2-CH
2-CH
2-,-CH
2-O-CH
2-,-CH
2-S-CH
2-,-CH
2-NH-CH
2-,-CH
2-N (CH
3)-CH
2-, and
X representes N-NO
2, N-CN or CH-NO
2
In each case, if maybe, saturated or unsaturated alkyl (for example alkyl or alkenyl) can be straight chain or side chain, it comprises with heteroatoms and makes up for example alkoxyl group.
These compounds are known to have insecticidal activity (consulting for example EP-A1-192 606, EP-A2-580 533, EP-A2-376 279, EP-A2-235 725).
The compound of the formula (I) that can be mentioned is " The Pesticide Manual ", the 13rd edition, and listed anabasine among 2003 (the British Crop Protection Council).
A kind of compound is for for example from EPAl 0 192 060 known formulas
Provado.
Another kind of compound is for for example from EPA2 0 302 389 known formulas
Ti304.
Another kind of compound is from the for example known formula of WO A191/04965
Acetamiprid.
Another kind of compound is for for example from EPA2 0 235 725 known formulas
Thiophene worm quinoline.
Another kind of compound is for example from EPA2 0 580 553 known formulas
Thiophene worm piperazine.
Another kind of compound is for example from EPA2 0 376 279 known formulas
Clothianadin.
Another kind of compound is for example from EPAl 0 649 845 known formulas
MTI-446.
Being specially adapted to of the present invention is the compound of formula (2), wherein substituting group " Het " expression chloropyridine base, for example Provado, Ti304, acetamiprid and thiophene worm quinoline
Preferred especially compound is Provado and thiophene worm quinoline.
In the degraded of the above-mentioned anabasine (for example Provado, Ti304 and thiophene worm quinoline) that has a 6-chlorine apellagrin group, the 6-chlorine apellagrin can be released.For example, Provado progressively is degraded to primary metabolite 6-chlorine apellagrin, and it is degraded to carbonic acid gas at last.Find that this metabolite also promotes plant or vegetable cell or grows up to the stress tolerance and the health of the seed of this type of plant, said plant or vegetable cell or the seed that grows up to this type of plant are transform as anti-coercing, and also can use according to the method for the invention.
Confirm that whether above-mentioned anabasine discharges 6-CNA when degrading in plant or in the specified plant a kind of method describes (Pflanzenschutz-Nachrichten Bayer46/1993,2 109-182) by Placke and Weber.
Therefore; In another embodiment of the present invention; Described and can be used for improving vegetable cell or plant or its part method to hypoxemia or oxygen free condition tolerance, said method comprises step: to said plant, the formula (3) of significant quantity is provided to vegetable cell or to the seed that grows up to said plant
6-chlorine apellagrin (nicotinic acid, CAS NO:5326-23-8).
Can come to provide the 6-chlorine apellagrin of significant quantity through the compound of directly using formula (3) to vegetable cell, plant, seed and/or its habitat to vegetable cell, plant or seed.Yet, also can to plant 6-CNA be provided through compound (for example above-claimed cpd) is provided, said compound can be produced metabolite 6-CNA by plant metabolism.
Should understand at once that above-claimed cpd also can be used to improve roots of plants and penetrate in growth medium or the soil; It comprises step: the anabasine compound (anabasine compound that for example comprises the formula (I) of chloropyridine side chain that the formula (I) of significant quantity is provided to vegetable cell, plant or seed or to the habitat of plant or to growth medium; Particularly can in plant, be produced 6-can by metabolism; The anabasine compound that comprises the formula (I) of chloropyridine side chain; Comprise Provado or thiophene worm quinoline), or 6-CNA is provided to vegetable cell, plant or seed or to the habitat of plant or to growth medium.
One of advantage of the present invention is The compounds of this invention and comprises the general characteristic of said compound compositions, and the plant in promptly just enough promoting to sprout with the seeds of these compositions-treated plants extends with the root of sprouting the plant that the back produces.
In another embodiment of the present invention; The method that promotes the roots of plants elongation that is applicable to has been described; It comprises that said compsn comprises the compound of formula (I) to said plant and/or its habitat, to vegetable cell or grow up to the compsn of the seed application significant quantity of said plant.
Therefore, the invention still further relates to compsn, said compsn comprises formula (I) compound that is used for this based composition purposes of the present invention.
The compound of formula (I) also can be used for the mixture with other active compounds (for example commercial useful dosage form or from the sterilant of the application form of this type formulation preparation, bactericide, miticide, mycocide etc.).Can mix to obtain the combinations thing, said compsn also resists the insect that possibly exist except promote the roots of plants elongation according to the present invention.Operable sterilant is for example organic phosphate agent, carbamate agent, carboxylate class chemical, chlorinated hydrocarbons chemical, the insect killing substance through microorganisms etc.
In many cases, this causes synergistic effect, and promptly the activity of mixture exceeds the activity of single composition.This type preparation and application form are useful especially on commercial and ecology, because can use the activeconstituents of lower amount usually.But synergistic agent itself must not have activity, as long as the effect that it can the enhanced activity compound.
With other known activity compounds (for example weedicide) or with the mixture of safener, fertilizer and growth regulator also be possible.
The processing of plant and plant part is directly carried out with active compound according to the present invention; Or allow compound that its periphery, environment or storage area are worked through conventional treatment method; Said conventional treatment method is for example through immersing, spray, evaporate, atomize, disperse, smear and also passing through to use one or more layers coating in (particularly under the situation at seed) under the situation of reproductive material.
Active compound can be converted into conventional formulation; For example solution, emulsion, wettable powders, suspensoid, pulvis, dust, paste, solubility powder, granule, suspension-s-emulsion enriched material, flooded the natural and synthetic materials of active compound and the microcapsule in the polymer material.
The content of active compound of the present invention in useful preparation of commerce or application form can change in wide in range scope.Can in wide in range qualification, change from the active compound content of the type of service of commercial formulation preparation.
These preparations provide in known manner, for example through active compound is mixed with supplement (being liquid solvent and/or solid carrier), randomly use tensio-active agent (being emulsifying agent and/or dispersion agent) and foam agent.
If the supplement that use are water, for example also possibly use organic solvent as solubility promoter.In essence, suitable liquid solvent is: perfume compound (like YLENE, toluene or alkylnaphthalene), chlorating perfume compound or chlorating aliphatic hydrocarbon such as chlorobenzene, vinylchlorid or methylene dichloride; Aliphatic hydrocarbon such as hexanaphthene or paraffin, for example petroleum fractions, mineral substance and vegetables oil; Alcohol is like butanols or glycol and ether and ester; Ketone such as acetone, methyl ethyl ketone, MIBK or pimelinketone; Intensive polar solvent such as N and DMSO 99.8MIN., and water.
Suitable solid carrier is: for example ammonium salt and ground natural mineral matter such as kaolin, clay, talcum, chalk, quartz, aminanthine, polynite or zeyssatite, and silicon-dioxide, aluminum oxide and the silicate of ground synthetic mineral matter such as high dispersing; Being used for the suitable solid carrier of particulate is: for example crushed and fractionated natural stone such as calcite, marble, float stone, sepiolite and rhombspar; The synthetic particle that also has inorganic and organic powder, and the particle of organic materials (like sawdust, coconut husk, corn cob and tobacco stem); Suitable emulsifying agent and/or foam agent are: for example non-ionic and anionic emulsifying agent such as polyoxyethylene fatty acid ester, polyoxyethylene aliphatic alcohol ether be alkylaryl polyglycol ether, AS, alkyl sulfuric ester, aromatic yl sulphonate for example, also has protein hydrolyzate; Suitable dispersion agent is: for example lignin sulphite waste liquid and methylcellulose gum.
Can use tackifier in the preparation, like natural and synthetic polymkeric substance (like Sudan Gum-arabic, Z 150PH and Yodo Sol VC 400) and the natural phospholipid (like kephalin and Yelkin TTS) and the synthetic phosphatide of CMC 99.5 and powder, particle or emulsion form.Other additives can be mineral substance and vegetables oil.
Possibly use tinting material such as inorganic pigment (for example red stone, titanium oxide and Prussian blue) and organic dye (like alizarine dyestuff, azoic dyestuff and metal phthalocyanine dyestuff), and micronutrient (like the salt of iron, manganese, boron, copper, cobalt, molybdenum and zinc).
Preparation comprises the active compound between 0.1wt% and the 98wt% usually, preferably between 0.1wt% and 90wt%, particularly preferably in the active compound between 0.5wt% and the 70wt%.
Under some utility ratio, anabasine compound and 6-CNA are significantly strong especially to the influence of the root growth degree of depth.Yet the utility ratio of active compound can change in wide in range relatively scope.Usually utility ratio for per hectare 1g to the 1600g active compound, preferred per hectare 10g is to the 800g active compound, especially preferably per hectare 10g is to the 600g active compound.
Of preamble; The present invention relates to be applicable to promote the roots of plants elongation to get in the growth medium or improve method that said method comprises and comprises formula (I) compound compositions to what plant propagation material (comprising the seed that grows up to plant) used significant quantity to the hypoxia condition tolerance.Can for example can handle (dress) seed prior to seeding at the reproductive material of plantation pre-treatment plant.Compound of the present invention also can be used on the seed grain through following manner: encapsulate seed grain with liquid preparation dipping seed grain or with solid preparation.When reproductive material is planted, for example at seeding time, also can compsn be applied to plant the position.
With regard to treatment of plant propagation material (like seed), the utility ratio of preference is generally the pending material of every 100kg with 0.1 to 1000g, particularly 1 arrives 800g, one of preferred 10 to 500g anabasine compound or 6-CNA.
All plants and plant part can be processed according to the present invention.Plant part is appreciated that all ground and underground parts, and plant organ (like branch, leaf, Hua Hegen), and the instance that can mention is leaf, needle, handle, stem, flower, sporophore, fruit, seed, root, stem tuber and rhizome.Plant part also comprises the material of results and nourish and generate material and sexual propagation material, for example cutting, stem tuber, rhizome, short crawl stem and seed.They also comprise vegetable cell, for example can be used for or derive from according to the present invention the conversion of vegetable cell.Also possibly be applied on the soil above-claimed cpd or in the soil; For example before plantation or sowing, use to reach said effect; The stress tolerance of the plant after for example enhancing is planted and the plant of sprouting, the plant of said sprouting are from being advanced the seed growth the treated soil by sowing.
Should understand immediately that also method of the present invention (comprise and use stress tolerance to strengthen transgenic or stress tolerance augment endogenous variant) can make up with method of the present invention (comprise and use anabasine compound or 6-CNA); Improving aspect hypoxemia or the oxygen free condition tolerance, or in improving growth medium or soil roots of plants dark aspect produce extra and collaborative effect.
Method of the present invention can be suitable for any plant---dicotyledonous and monocotyledons; Include but not limited to cotton, Btassica (Brassica) plant, colea, wheat, cereal or corn, barley, Sunflower Receptacle, rice, oat, sugarcane, soybean, vegetables (comprising witloof, lettuce, tomato), tobacco, yam, beet, pawpaw, pineapple, mango, Arabidopis thaliana; But also comprise the plant that is used for gardening, flowers or forestry, the cereal plant comprises wheat, oat, barley, rye, rice, turfgrass, Chinese sorghum, grain or sugarcane plants.Method of the present invention also can be used for any plant, includes but are not limited to cotton, tobacco, rape, colea, soybean, vegetables, yam, Lemna species (Lemna spp.), Nicotiana species (Nicotiana spp.), sweet potato, Arabidopis thaliana, clover, barley, beans, cereal, cotton, flax, pea, rape, rice, rye, safflower, Chinese sorghum, soybean, Sunflower Receptacle, tobacco, wheat, Asparagus, beet, asparagus broccoli, Caulis et Folium Brassicae capitatae, Radix Dauci Sativae, Cauliflower, celery, cucumber, eggplant, lettuce, onion, colea, pepper, yam, summer squash, Rhaphanus, spinach genus, pumpkin, tomato, american pumpkin (zucchini), almond, apple, apricot genus, banana, blackberry, blueberry, genus vaccinium, cocoa tree, cherry, coconut, big fruit blueberry, nipa palm, Vitis, natsudaidai, Psidium, macaque peach, lemon, comes lemon, mango, melon (melon), nectarine, orange, papaya genus, passion fruit, peach, peanut, pears, pineapple, Pistacia vera (pistachio), plum (plum), raspberry (raspberry), strawberry, oranges and tangerines, English walnut and watermelon.
" comprising " that this paper is used is interpreted as the existence of the said characteristic that is related to of explanation, integral body, step or composition, but do not get rid of existence or add one or more characteristics, integral body, step or composition or its set.Therefore, the nucleic acid or the protein that for example comprise Nucleotide or aminoacid sequence can comprise more polynucleotide or amino acid except that Nucleotide of in fact quoting or amino acid, promptly are arranged in bigger nucleic acid or protein.The transgenic that comprises the DNA district that defines on function or the structure can comprise extra DNA district etc.
Only if explanation is arranged in an embodiment in addition; All recombinant DNA technology bases are like people such as Sambrook (1989) Molecular Cloning:A Laboratory Manual; Second edition, ColdSpring Harbor Laboratory Press, people such as NY and Ausubel (1994) CurrentProtocols in Molecular Biology; Current Protocols, the 1st volume of the U.S. and the standard scheme described in the 2nd volume are accomplished.The standard material and the method that are used for the plant molecular operation are described in Plant Molecular Biology Labfax (1993) by R.D.D.Croy; It is by BIOS ScientificPublications Ltd (Britain) and Blackwell Scientific Publications, Britain's combined publication.Other bibliographys that are used for standard molecular biological technique comprise Sambrook and Russell (2001) Molecular Cloning:A Laboratory Manual; The third edition; Cold Spring Harbor Laboratory Press, NY, Brown (1998) Molecular Biology LabFax; Second edition, the I volume of Academic Press (Britain) and II volume.The standard material and the method that are used for the polymerase chain reaction are found in Dieffenbach and Dveksler (1995) PCR Primer:ALaboratory Manual; People (2000) PCR-Basics:From Background to Bench such as Cold Spring Harbor Laboratory Press and McPherson; First version; SpringerVerlag, Germany.
The following sequence of reference in specification sheets and embodiment full text:
SEQ ID No.1: from the parp1 coding region of Arabidopis thaliana
SEQ ID No.2: from the parp2 coding region of Arabidopis thaliana
SEQ ID No.3: from the parp1 coding region 1 of corn
SEQ ID No.4: from the parp1 coding region 2 of corn
SEQ ID No.5: from the parp2 coding region of corn
SEQ ID No.6: from the parp2 part coding region of cotton
SEQ ID No.7: from the parG coding region of Arabidopis thaliana
SEQ ID No.8: from the parG coding region of yam (Solanum tuberosum)
SEQ ID No.9: from the parG coding region of rice
SEQ ID No.10: from the parG coding region of corn
SEQ ID No.11: from the nucleotide sequence of the nicotinamidase (PNC1) of yeast saccharomyces cerevisiae
SEQ ID No.12: from the nucleotide sequence (complementary sequence) of the nicotinate/ester phosphoribosyl transferase (NPT1) of yeast saccharomyces cerevisiae
SEQ ID No.13: from the nucleotide sequence of the NAMN adeninyl transferring enzyme 1 (NMA1) of yeast saccharomyces cerevisiae
SEQ ID No.14: from the nucleotide sequence of the NAMN adeninyl transferring enzyme 2 (NMA2) of yeast saccharomyces cerevisiae
SEQ ID No.15: from the nucleotide sequence of the NAD synthetic enzyme (QNS1) of yeast saccharomyces cerevisiae
SEQ ID No.16: from the nucleotide sequence (isotype 1) of the nicotinamidase of Arabidopis thaliana
SEQ ID No.17: from the nucleotide sequence (isotype 2) of the nicotinamidase of Arabidopis thaliana
SEQ ID No.18: from the nucleotide sequence (isotype 3) of the nicotinamidase of Arabidopis thaliana
SEQ ID No.19: from the nucleotide sequence (isotype 1) of the nicotinate/ester phosphoribosyl transferase of Arabidopis thaliana
SEQ ID No.20: from the nucleotide sequence (isotype 2) of the nicotinate/ester phosphoribosyl transferase of Arabidopis thaliana
SEQ ID No.21: from the nucleotide sequence of the NAMN adeninyl transferring enzyme of Arabidopis thaliana
SEQ ID No.22: from the nucleotide sequence of the NAD synthetic enzyme of Arabidopis thaliana
Embodiment
Embodiment 1: the scheme that is used for measuring the growth medium Arabidopis thaliana root growth degree of depth
Substratum:
Germination medium: the Murashige of a half strength and Skoog salt; Vitamin B5; 1.5% sucrose; PH 5.8; 0.4%Difco agar.
Arabidopsis thaliana
Arabidopis thaliana seed sterilization: 70% ethanol 2 minutes; SYNTHETIC OPTICAL WHITNER (6% reactive chlorine)+1 Tween 20 was made into 20ml solution 10 minutes; With aseptic tap water washing 5 times; In the 2ml Eppendorf tube, sterilize.The Arabidopis thaliana seed sinks to the pipe end, allows to remove liquid by means of the 1ml autospencer.
Seed is sprouted in advance: the low light level spent the night to 24 hours in containing 9 centimetres of Optilux petridish (Falcon) of the aseptic tap water of 10ml.
Arabidopsis thaliana growth: with planting seed in contain ± 25 * 150 millimeters Glass tubings (Sigma C5916) with natural (transparent) coloured lid (Sigma C5791) of 34ml germination medium in: 1 seed/pipe.Place test-tube stand (VWR nalg5970-0025) outside two of 40 pipes to go pipe, wrap with aluminium foil and make that root can growth in darkness.Plant is 23 ℃ of cultivations.30-50μEinstein?s
-1m
-2。12 hours illumination-12 hour dark.(see figure 1).
The measurement root is dark
After three weeks, it is dark that the deepest point from the media surface to the root growth is measured root.(see figure 1).
Embodiment 2: comprised the analysis that strengthens the genetically modified arabidopsis thaliana root growth degree of depth of stress tolerance
Described in embodiment 1, cultivate the genetically modified arabidopsis thaliana that comprises described in WO 00/04173A1 (for example in embodiment 8), said transgenes encoding dsRNA molecule, said dsRNA molecule can reduce endogenous PARP1 or PARP2 expression of gene.
After three weeks, the root of measuring transgenic plant compares deeply and with non-transgenic control plant of cultivating in a similar manner or the root of the homogenic plant of non-transgenic deeply.
In first experiment; To comprise multiple population of genetically modified Arabidopis thaliana cv.Col-0 plant and the non-transgenic population of non-transgenic Arabidopis thaliana cv.Col-0 plant compares; Said transgenes encoding dsRNA molecule; Said dsRNA molecule can reduce endogenous PARP2 expression of gene (strain is that 427-16 and 427-20 show the weak tolerance that high light is coerced, and strain is that 427-19 shows the medium tolerance that high light is coerced).
Measuring result is carried out statistical analysis, be summarised in the table 1, table 1 has also been represented average, standard deviation and fiducial interval.
Compare with non-transgenic Arabidopis thaliana cv.Col-0 control plant, the root statistics that the high light stress conditions is had a transgenic arabidopsis plant (strain is 427-19) of height endurability stretches in the growth medium darker (99% confidence level) (seeing Fig. 2 and table 1) significantly.
Table 1: compare with non-transgenic Arabidopis thaliana cv.Col-0 plant, comprise the root dark (mm) of genetically modified Arabidopis thaliana cv.Col-0 plant, said transgenes encoding dsRNA molecule, said dsRNA molecule can reduce endogenous PARP2 expression of gene
| ? | Col-0 | 427-16 | 427-19 | 427-20 |
| Average | 25.173611 | 26.388889 | 27.42 | 24.942029 |
| Standard deviation | 3.64166 | 3.422548 | 3.361185 | 3.69174 |
| Standard error | 0.429174 | 0.46575 | 0.388116 | 0.444433 |
| 95% puts letter | 0.858348 | 0.94128 | 0.776233 | 0.888866 |
| 99% puts letter | 1.141602 | 1.259388 | 1.032389＊ | 1.182192 |
＊p＜0.01
In another embodiment; To comprise transgenic and high light coerced tolerance transgenic arabidopsis cv.Col-0 plant population (strain is 427-22) with contain similar transgenic but high light coerced responsive transgenic line (strain is 427-24); And non-transgenic Arabidopis thaliana cv.Col-0 control plant relatively; Wherein said transgenes encoding dsRNA molecule, said dsRNA molecule can reduce endogenous PARP2 expression of gene.
Measuring result is carried out statistical analysis, be summarised in the table 2, table 2 has also been represented average, standard deviation and fiducial interval.
Compare with the root of coercing responsive transgenic arabidopsis Col-0 plant strain system (strain is 427-24) with the root of Arabidopis thaliana cv.Col-0 control plant, the root of the transgenic arabidopsis cv.Col-0 plant of the transgenic line of stress-tolerance (strain is 427-22) stretches in the growth medium Difco agar of (comprise 0.7% (replacing 0.4%) and) darker (seeing Fig. 3 and table 2).
Table 2: compare with non-transgenic Arabidopis thaliana cv.Col-0 plant, comprise the root dark (mm) of genetically modified Arabidopis thaliana cv.Col-0 plant, said transgenes encoding dsRNA molecule, said dsRNA molecule can reduce endogenous PARP2 expression of gene
| ? | Col-0 | 427-22 | 427-24 |
| Average | 18.291339 | 21.146154 | 17.384058 |
| Standard deviation | 2.928677 | 5.368981 | 2.740149 |
| Standard error | 0.259878 | 0.66594 | 0.329875 |
| 95% puts letter | 0.514558 | 1.33188 | 0.65975 |
| 99% puts letter | 0.680101 | 1.771401＊ | 0.877468 |
＊p＜0.01
In another embodiment, analyze following population:
C24: wild-type Arabidopis thaliana strain system; Strain is 1599: comprise the genetically modified Arabidopis thaliana transgenic line that the high light stress conditions is had height endurability of anti--PARP2; Strain is 1463: comprise the genetically modified Arabidopis thaliana transgenic line that the high light stress conditions is had medium tolerance of anti--PARP2; Strain is 1681: the Arabidopis thaliana transgenic line that the high light stress conditions is had medium tolerance that comprises anti--PARP1 gene; With strain be 1690: the Arabidopis thaliana transgenic line that the high light stress conditions is had medium tolerance that comprises anti--PARP1 gene.Strain is that 1599 stress tolerance is very high, and strain is that 1463,1681 and 1690 stress tolerance is therefrom waited until high variation.
Measuring result is carried out statistical analysis, and be summarized in the table 3, table 3 expression average, standard deviation and fiducial interval.
Compare with non-transgenic Arabidopis thaliana cv.C24 control plant; The root that comprises genetically modified transgenic arabidopsis cv.C24 plant stretches in the growth medium darker; Wherein said transgenes encoding dsRNA molecule, said dsRNA molecule can reduce the expression (seeing Fig. 4 and table 3) of endogenous PARP1 gene (strain is 1681 and 1690) or PARP2 gene (strain is 1599 and 1463).
Table 3: compare with non-transgenic Arabidopis thaliana cv.C24 plant, comprise the root dark (mm) of genetically modified Arabidopis thaliana cv.C24 plant, said transgenes encoding dsRNA molecule, said dsRNA molecule can reduce endogenous PARP1 or PARP2 expression of gene
| ? | |
1599 | 1463 | 1681 | 1690 |
| Average | 16.18 | 18.125 | 17.405063 | 17.433333 | 17.897727 |
| Standard deviation | 2.192181 | 2.992481 | 2.16036 | 2.112246 | 1.885007 |
| Standard error | 0.219218 | 0.451133 | 0.243059 | 0.27269 | 0.284175 |
| 95% puts letter | 0.438436 | 0.911741 | 0.486119 | 0.551106 | 0.574319 |
| 99% puts letter | 0.58312 | 1.219865＊ | 0.646538＊ | 0.737353＊ | 0.76841＊ |
＊p＜0.01
In another embodiment; Analysis comprises transgenic and high light is coerced 1: 1 segregating population of the transgenic arabidopsis cv.Col-0 strain system with high resistance; Wherein said transgenes encoding dsRNA molecule, said dsRNA molecule can reduce endogenous PARP2 expression of gene.Genetically modified existence confirms through pcr analysis.
Compare with the azygous Arabidopis thaliana cv.Col-0 plant that from strain is 427-19, comprise genetically modified plant and have and stretch into root darker in the growth medium (seeing Fig. 5 and table 4).
Table 4: compare with azygous Arabidopis thaliana cv.Col-0 plant, comprise the root dark (mm) of genetically modified Arabidopis thaliana cv.Col-0 plant, said transgenes encoding dsRNA molecule, said dsRNA molecule can reduce endogenous PARP2 expression of gene
| ? | Azygous 427-19 | Genetically modified 427-19 |
| Average | 25.296512 | 27.38172 |
| Standard deviation | 3.573907 | 3.244894 |
| Standard error | 0.385384 | 0.33648 |
[0259]
| 95% puts letter | 0.770769 | 0.67296 |
| 99% puts letter | 1.025122 | 0.895036* |
＊p＜0.01
Embodiment 3: the analysis of the arabidopsis thaliana root growth degree of depth behind the application Provado
Said like embodiment 1 with the cultivation on the germination medium that comprises multiple concentration Provado (0,50 and 100 mg/litre) (the Difco agar that contains 0.7% (replacing 0.4%)) of Arabidopis thaliana cv.C24 plant.The degree of depth with the roots of plants of 50 and 100 mg/litre Provados processing is measured in three week backs, and with the depth ratio of the root of the plant of being untreated of cultivation in the same manner.
Compare with the root of untreated arabidopsis thaliana, the root of treated arabidopsis thaliana stretches in the growth medium darker (seeing Fig. 6 and table 5).
Table 5: do not compare with the Arabidopis thaliana cv.C24 plant of handling, with the root of the Arabidopis thaliana cv.C24 plant of 50 and 100 mg/litre Provados processing (mm) deeply with Provado
| ? | 0 mg/litre | 50 mg/litre | 100 mg/litre |
| Average | 1.9475 | 2.1875 | 2.2725 |
| Standard deviation | 0.289714 | 0.353508 | 0.456072 |
| Standard error | 0.037402 | 0.045638 | 0.058879 |
| 95% puts letter | 0.075589 | 0.092234 | 0.118994 |
| 99% puts letter | 0.101135 | 0.123404＊ | 0.159208＊ |
＊p＜0.01
Embodiment 4: the analysis of using 6-chlorine apellagrin (6-CNA) the back arabidopsis thaliana root growth degree of depth
As embodiment 1 is said Arabidopis thaliana cv.C24 plant is cultivated on the germination medium that comprises multiple concentration 6-CNA (0,1 and 5 mg/litre).The degree of depth of the roots of plants of handling with 1 and 5 mg/litre 6-CNA is measured in three week backs, and with cultivation in the same manner not with the depth ratio of the root of the plant of 6-CNA processing.
Compare with the root of untreated arabidopsis thaliana, the root of treated plant stretches in the growth medium darker (seeing Fig. 7 and table 6).
Table 6: do not compare with the Arabidopis thaliana cv.Col-0 plant of handling, with the root of the Arabidopis thaliana cv.C24 plant of 1 and 5 mg/litre 6-CNA processing (mm) deeply with 6-CNA
| ? | 0 mg/ |
1 mg/ |
5 mg/litre |
| Average | 2.031034 | 2.179661 | 2.118367 |
| Standard deviation | 0.189373 | 0.186418 | 0.237762 |
| Standard error | 0.024866 | 0.02427 | 0.033966 |
| 95% puts letter | 0.050254 | 0.049049 | 0.068645＊＊ |
| 99% puts letter | 0.067237 | 0.065625＊ | 0.091844 |
＊p＜0.01
＊＊p＜0.05。
Claims (27)
1. be used to improve vegetable cell or the plant method to hypoxemia or oxygen free condition tolerance, it comprises step:
A) cell to said vegetable cell or said plant provides stress tolerance to strengthen transgenic; Wherein said stress tolerance strengthens transgenic and is selected from:
The stress tolerance that i. can reduce plant endogenous PARP genetic expression strengthens transgenic, particularly wherein said transgenes encoding PARP inhibitory RNA molecules;
The stress tolerance that ii. can reduce plant endogenous PARG genetic expression strengthens transgenic, particularly wherein said transgenes encoding PARG inhibitory RNA molecules; Or
Iii. the stress tolerance of plant function enzyme of Reduced nicotinamide-adenine dinucleotide salvage route of encoding strengthens transgenic, and said enzyme is selected from nicotinamidase, nicotinate/ester phosphoribosyl transferase, NAMN adeninyl transferring enzyme or Reduced nicotinamide-adenine dinucleotide synthetic enzyme.
2. be used to promote that roots of plants penetrates into the method for growth medium, it comprises step:
A) cell to said vegetable cell or said plant provides stress tolerance to strengthen transgenic; Wherein said stress tolerance strengthens transgenic and is selected from:
The stress tolerance that i. can reduce plant endogenous PARP genetic expression strengthens transgenic, particularly wherein said transgenes encoding PARP inhibitory RNA molecules;
The stress tolerance that ii. can reduce plant endogenous PARG genetic expression strengthens transgenic, particularly wherein said transgenes encoding PARG inhibitory RNA molecules; Or
Iii. the stress tolerance of plant function enzyme of Reduced nicotinamide-adenine dinucleotide salvage route of encoding strengthens transgenic, and said enzyme is selected from nicotinamidase, nicotinate/ester phosphoribosyl transferase, NAMN adeninyl transferring enzyme or Reduced nicotinamide-adenine dinucleotide synthetic enzyme.
3. according to the method for claim 1 or 2, wherein said stress tolerance enhancing gene coding PARP inhibitory RNA molecules.
4. according to the method for claim 3, wherein said transgenic comprises the dna fragmentation of following effective connection:
A) the effable promotor of plant;
B) the DNA district of coding PARP inhibitory RNA molecules, it comprises at least 19 in 20 continuous nucleotides of nucleotide sequence of nucleotide sequence or SEQ ID No 6 of nucleotide sequence, SEQ ID No 5 of nucleotide sequence, the SEQ ID No 4 of nucleotide sequence, the SEQ ID No 3 of the nucleotide sequence that is selected from SEQ ID No 1, SEQ ID No 2; With
C) Transcription Termination and polyadenylic acid DNA district.
5. according to the method for claim 3, wherein said transgenic comprises the dna fragmentation of following effective connection:
A) the effable promotor of plant;
B) the DNA district of coding PARP inhibitory RNA molecules, it comprises at least 19 in 20 continuous nucleotides of complementary sequence of nucleotide sequence of nucleotide sequence or SEQ ID No 6 of nucleotide sequence, SEQ ID No 5 of nucleotide sequence, the SEQ ID No 4 of nucleotide sequence, the SEQ ID No 3 of the nucleotide sequence that is selected from SEQ ID No 1, SEQ ID No 2; With
C) Transcription Termination and polyadenylic acid DNA district.
6. according to the method for claim 3, wherein said transgenic comprises the dna fragmentation of following effective connection:
A) the effable promotor of plant;
B) the DNA district of coding PARP inhibitory RNA molecules, said RNA molecule comprises:
I., sense nucleotide sequence is arranged, and it comprises at least 19 in 20 continuous nucleotides of nucleotide sequence of nucleotide sequence or SEQ ID No 6 of nucleotide sequence, SEQ ID No 5 of nucleotide sequence, the SEQ ID No 4 of nucleotide sequence, the SEQ ID No 3 of the nucleotide sequence that is selected from SEQ ID No 1, SEQ IDNo 2; With
Ii. antisense base sequences, it comprises with said has at least 20 continuous nucleotide complementary nucleotide sequences described in the sense nucleotide sequence,
Wherein said have justice and antisense base sequences can form the double-stranded RNA district; And
C) Transcription Termination and polyadenylic acid DNA district.
7. according to the method for claim 6, wherein said antisense base sequences and said has sense nucleotide sequence to have about 95% sequence identity or identical.
8. according to the method for claim 1 or claim 2, wherein said transgenes encoding ParG inhibitory RNA molecules.
9. according to Claim 8 method, wherein said transgenic comprises the dna fragmentation of following effective connection:
A) the effable promotor of plant;
B) the DNA district of coding PARG inhibitory RNA molecules, it comprises at least 19 in 20 continuous nucleotides of nucleotide sequence of nucleotide sequence or SEQ ID No 10 of nucleotide sequence, SEQ ID No 9 of the nucleotide sequence that is selected from SEQ ID No7, SEQ ID No 8; With
C) Transcription Termination and polyadenylic acid DNA district.
10. according to Claim 8 method, wherein said transgenic comprises the dna fragmentation of following effective connection:
A) the effable promotor of plant;
B) the DNA district of coding PARG inhibitory RNA molecules, it comprises at least 19 in 20 continuous nucleotides of complementary sequence of nucleotide sequence of nucleotide sequence or SEQ ID No 10 of nucleotide sequence, SEQ ID No 9 of the nucleotide sequence that is selected from SEQ ID No7, SEQ ID No 8; With
C) Transcription Termination and polyadenylic acid DNA district.
11. method according to Claim 8, wherein said transgenic comprises the dna fragmentation of following effective connection:
A) the effable promotor of plant;
B) the DNA district of coding PARG inhibitory RNA molecules, said RNA molecule comprises:
I., sense nucleotide sequence is arranged, and it comprises at least 19 in 20 continuous nucleotides of nucleotide sequence of nucleotide sequence or SEQ ID No 10 of nucleotide sequence, SEQ ID No 9 of the nucleotide sequence that is selected from SEQ ID No 7, SEQ IDNo 8; With
Ii. antisense base sequences, it comprises with said has at least 20 continuous nucleotide complementary nucleotide sequences described in the sense nucleotide sequence,
Wherein said have justice and antisense base sequences can form the double-stranded RNA district; With
C) Transcription Termination and polyadenylic acid DNA district.
12. according to the method for claim 11, wherein said antisense base sequences and said has sense nucleotide sequence to have about 95% sequence identity or identical.
13. method according to claim 1 or claim 2; The plant function enzyme of wherein said transgenes encoding Reduced nicotinamide-adenine dinucleotide salvage route, said enzyme are selected from nicotinamidase, nicotinate/ester phosphoribosyl transferase, NAMN adeninyl transferring enzyme or Reduced nicotinamide-adenine dinucleotide synthetic enzyme.
14. method according to claim 13; Wherein said transgenic comprises following nucleotide sequence, and said nucleotide sequence is selected from the nucleotide sequence of SEQ ID 11, the nucleotide sequence of SEQ ID 12, the nucleotide sequence of SEQ ID 13, the nucleotide sequence of SEQ ID 14, the nucleotide sequence of SEQ ID 15, the nucleotide sequence of SEQ ID 16, the nucleotide sequence of SEQ ID 17, the nucleotide sequence of SEQ ID 18, the nucleotide sequence of SEQ ID 19, the nucleotide sequence of SEQ ID 20, the nucleotide sequence of SEQID 21 or the nucleotide sequence of SEQ ID 22.
15. according to each method in the claim 1 to 14, it comprises another step, promptly to said plant or its habitat, or the seed of said plant is used formula (I) compound of significant quantity
Wherein
Het representes heterocycle, and said heterocycle is optional in each case by fluorine, chlorine, methyl or ethyl list replacement or polysubstituted, and said heterocycle is selected from down the group heterocycle:
Pyridin-3-yl, pyridine-5-base, 3-pyridyl, 1-oxidation-5-pyridyl, THF-3-base, thiazole-5-base,
A representes C
1-C
6-alkyl ,-N (R
1) (R
2) or S (R
2),
Wherein
R
1Expression hydrogen, C
1-C
6-alkyl, phenyl-C
1-C
4-alkyl, C
3-C
6-naphthenic base, C
2-C
6-alkenyl or C
2-C
6-alkynyl, and
R
2Expression C
1-C
6-alkyl, C
2-C
6-alkenyl, C
2-C
6-alkynyl ,-C (=O)-CH
3Or benzyl,
R representes hydrogen, C
1-C
6-alkyl, C
2-C
6-alkenyl, C
2-C
6-alkynyl ,-C (=O)-CH
3Or benzyl, or and R
2Represent following group together:
-CH
2-CH
2-,-CH
2-CH
2-CH
2-,-CH
2-O-CH
2-,-CH
2-S-CH
2-,-CH
2-NH-CH
2-,-CH
2-N (CH
3)-CH
2-, and
X representes N-NO
2, N-CN or CH-NO
2
16. according to the method for claim 15, the said heterocycle representative of being represented by Het in wherein said formula (I) compound is by the substituted pyridin-3-yl heterocycle of chlorine.
17. the method for claim 16, the compound of wherein said (I) formula are Provado or thiophene worm quinoline.
18. according to each method in the claim 1 to 14, it also comprises step: the cell that the 6-chlorine apellagrin of significant quantity is offered said plant.
19. be used to improve vegetable cell or the plant method to hypoxemia or oxygen free condition tolerance, it comprises step: the 6-chlorine apellagrin of significant quantity is offered said vegetable cell or plant.
20. be used to promote that roots of plants penetrates into the method for growth medium, it comprises step: the cell that the 6-chlorine apellagrin of significant quantity is offered said plant.
21. according to the method for claim 19 or claim 20, wherein through using formula (I) compound of significant quantity and said 6-chlorine apellagrin is provided to said vegetable cell or plant to said plant or its habitat or to the seed of said plant
Wherein
Het representes heterocycle, and said heterocycle is optional in each case by chlorine list replacement or polysubstituted, and said heterocycle is selected from down the group heterocycle: pyridin-3-yl or pyridine-5-base,
A representes C
1-C
6-alkyl ,-N (R
1) (R
2) or S (R
2),
Wherein
R
1Expression hydrogen, C
1-C
6-alkyl, phenyl-C
1-C
4-alkyl, C
3-C
6-naphthenic base, C
2-C
6-alkenyl or C
2-C
6-alkynyl, and
R
2Expression C
1-C
6-alkyl, C
2-C
6-alkenyl, C
2-C
6-alkynyl ,-C (=O)-CH
3Or benzyl,
R representes hydrogen, C
1-C
6-alkyl, C
2-C
6-alkenyl, C
2-C
6-alkynyl ,-C (=O)-CH
3Or benzyl, or and R
2Represent following group together:
-CH
2-CH
2-,-CH
2-CH
2-CH
2-,-CH
2-O-CH
2-,-CH
2-S-CH
2-,-CH
2-NH-CH
2-,-CH
2-N (CH
3)-CH
2-, and
X representes N-NO
2, N-CN or CH-NO
2
22. according to the method for claim 21, the anabasine that wherein comprises the chloropyridine side chain discharges said 6-chlorine apellagrin in the plant degradation process.
23. the method for claim 22, wherein said anabasine are Provado or thiophene worm quinoline.
24., wherein said 6-chlorine apellagrin is directly applied to said plant or its said habitat, said vegetable cell or said seed according to the method for claim 19 or claim 20.
25. following material is used for promoting that roots of plants stretches into the purposes of growth medium:
A) foreign DNA, it comprises stress tolerance and strengthens transgenic or strengthen the variant of genetically modified native gene corresponding to this type stress tolerance, and/or
B) 6-chlorine apellagrin or formula (I) compound
Wherein
Het representes heterocycle, and said heterocycle is optional in each case by fluorine, chlorine, methyl or ethyl list replacement or polysubstituted, and said heterocycle is selected from down the group heterocycle:
Pyridin-3-yl, pyridine-5-base, 3-pyridyl, 1-oxidation-5-pyridyl, THF-3-base, thiazole-5-base,
A representes C
1-C
6-alkyl ,-N (R
1) (R
2) or S (R
2),
Wherein
R
1Expression hydrogen, C
1-C
6-alkyl, phenyl-C
1-C
4-alkyl, C
3-C
6-naphthenic base, C
2-C
6-alkenyl or C
2-C
6-alkynyl, and
R
2Expression C
1-C
6-alkyl, C
2-C
6-alkenyl, C
2-C
6-alkynyl ,-C (=O)-CH
3Or benzyl,
R representes hydrogen, C
1-C
6-alkyl, C
2-C
6-alkenyl, C
2-C
6-alkynyl ,-C (=O)-CH
3Or benzyl, or and R
2Represent following group together:
-CH
2-CH
2-,-CH
2-CH
2-CH
2-,-CH
2-O-CH
2-,-CH
2-S-CH
2-,-CH
2-NH-CH
2-,-CH
2-N (CH
3)-CH
2-, and
X representes N-NO
2, N-CN or CH-NO
2
26. following material is used to improve the purposes of plant to hypoxemia or oxygen free condition tolerance:
A) foreign DNA, it comprises stress tolerance and strengthens transgenic or strengthen the variant of genetically modified native gene corresponding to this type stress tolerance, and/or
B) 6-chlorine apellagrin or formula (I) compound
Wherein
Het representes heterocycle, and said heterocycle is optional in each case by fluorine, chlorine, methyl or ethyl list replacement or polysubstituted, and said heterocycle is selected from down the group heterocycle:
Pyridin-3-yl, pyridine-5-base, 3-pyridyl, 1-oxidation-5-pyridyl, THF-3-base, thiazole-5-base,
A representes C
1-C
6-alkyl ,-N (R
1) (R
2) or S (R
2),
Wherein
R
1Expression hydrogen, C
1-C
6-alkyl, phenyl-C
1-C
4-alkyl, C
3-C
6-naphthenic base, C
2-C
6-alkenyl or C
2-C
6-alkynyl, and
R
2Expression C
1-C
6-alkyl, C
2-C
6-alkenyl, C
2-C
6-alkynyl ,-C (=O)-CH
3Or benzyl,
R representes hydrogen, C
1-C
6-alkyl, C
2-C
6-alkenyl, C
2-C
6-alkynyl ,-C (=O)-CH
3Or benzyl, or and R
2Represent following group together:
-CH
2-CH
2-,-CH
2-CH
2-CH
2-,-CH
2-O-CH
2-,-CH
2-S-CH
2-,-CH
2-NH-CH
2-,-CH
2-N (CH
3)-CH
2-, and
X representes N-NO
2, N-CN or CH-NO
2
27. the purposes of claim 26 wherein brings said plant through being exposed to waterlogging, submergence or flooding with said hypoxemia or oxygen free condition.
Applications Claiming Priority (4)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP05076392.9 | 2005-06-15 | ||
| EP05076392 | 2005-06-15 | ||
| US69110305P | 2005-06-16 | 2005-06-16 | |
| US60/691,103 | 2005-06-16 |
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|---|---|---|---|
| CN200680021382XA Division CN101218345B (en) | 2005-06-15 | 2006-06-06 | Methods for increasing the resistance of plants to hypoxic conditions |
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|---|---|---|---|
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