CN101443451A

CN101443451A - Novel stress-related microRNA molecules and uses thereof

Info

Publication number: CN101443451A
Application number: CNA200780017330XA
Authority: CN
Inventors: M·博茨; M·梅茨拉夫
Original assignee: Universiteit Gent; Vlaams Instituut voor Biotechnologie VIB; Bayer CropScience NV
Current assignee: Universiteit Gent; Vlaams Instituut voor Biotechnologie VIB; Bayer CropScience NV
Priority date: 2006-05-12
Filing date: 2007-05-10
Publication date: 2009-05-27
Also published as: BRPI0711658A2; ATE519852T1

Abstract

Naturally occurring and synthetic novel stress-related miRNAs are provided which can be used to modify the stress tolerance of plants.

Description

Novel stress-related microRNA molecules and uses thereof

Invention field

The present invention relates to agriculture field, more particularly, relate to novel stress-related microRNA molecules (miRNA) and be used to change plant to the tolerance of unfavorable growth conditions and be used to improve the purposes of plant the tolerance of stress conditions, described stress conditions is the inanimate stress conditions particularly, such as but not limited to low or high temperature, arid, highlight strength, chemical pollution, water logging, high salinity, highlight strength, high UV radiation etc.The method of novel miRNA described herein and precursor thereof and this type of miRNA of use and precursor thereof increases or reduces stress tolerance in plants.This paper has also described synthetic miRNA, is used to increase the stress tolerance of plant.

Background technology

Length is that the short rna molecule of about 21 to 24 Nucleotide has been presented at and relates to PTGS mechanism in the eukaryote.These molecules are (to be DICER in animal by RNAse III enzyme; DICERLIKE in plant) long double stranded rna molecule cutting is produced, they are used as the guide sequence that mixes in the RISC complex body, are used for the sequence-specific degraded to target RNA (mRNA) molecule.The short rna molecule also can be raised the silencing complex that into disturbs expression of target gene in the sequence-specific mode, and described interference is by specific chromatin reconstitution and methylate and realize.

Recently, found the short rna molecule of a class novelty in animal and plant, they are named as Microrna (miRNA).Microrna is little endogenous RNA, and they are not only at plant regulate gene expression in other eukaryote also.In plant, the RNA that these about 21 Nucleotide are grown processes from the stem ring zone of growing endogenous primary transcribe by the nicking activity of DCL1 to get.Mirnas of plant and conservative said target mrna height complementation, it instructs the cutting of their target.MiRNA seems that be key ingredient in to the regulation and control that relate to for example genetic expression of the complicated approach network of growth.

In animal, miRNA relates to two steps from the biology formation of long primary transcribe (pri-miRNA).At first, by on every arm of stem ring, cutting, the pri-miRNA from nuclear discharge miRNA stem ring (preceding-miRNA), this is realized by Drosha.After outputing to kytoplasm, DICER carries out the second cover cutting, so that (reverse complemental Microrna adjacent thereto forms duplex with miRNA ^*) separate with the ring of preceding-miRNA is regional.In plant, two steps are all undertaken by DCL1, and infer that two steps all take place in nuclear.

International Patent Application WO 03/029459 has been described the RNA of 22 and 21 Nucleotide that play a role as crucial regulon in growth opportunity of beautiful new rhabditis axei (Caenorhabditis elegans).

WO2004/009779 has described and has been used to regulate the nucleotide sequence expression, especially for the composition and the method for the expression of regulatory gene in plant.Composition comprises the precursor RNA construct that is used for the expressed rna precursor.The precursor RNA construct is included in expression promoter in the vegetable cell, and its driving has the expression of the precursor RNA of Microrna.The part of miRNA and target gene or target nucleotide sequences is complementation or part complementary, and it acts on regulates target sequence or target gene expression.By this way, RNA precursor construct can be designed to regulate the expression of any interested nucleotide sequence (endogenous plant gene or transgenosis).The precursor RNA construct can be used in combination with modulator, to strengthen the effect to genetic expression.When having precursor RNA, the expression of modulator changes the accumulation of miRNA, therefore strengthens the ability of regulation and control of miRNA.The document has also been described the purposes that modulator is expressed by siRNA and miRNA approach controlling gene.Also provide through plant transformed, tissue, cell and seed.

WO2005/017111 has described a kind of method, is used to identify the Microrna recognition component and produces Microrna, and it has also described system and the computer program that is used to carry out these class methods.It also discloses the recombinant nucleic acid molecules that comprises allogeneic coding sequence and one or more MRE, and disclose separated, comprise one or more MRE sequences and do not contain the nucleic acid molecule of the encoding sequence that effectively links to each other with controlling element.The purposes that Microrna that the method by this invention of also having described produces and described Microrna are used for down-regulation of gene expression.

WO 2005/035769 and WO 2005/052170 provide the method and composition that can be used for target sequence containment (suppression) and target sequence affirmation.This application has also been described the polynucleotide constructs that can be used for gene silencing, and the cell, the Plants and Seeds that comprise described polynucleotide.It also provides the method for Microrna with reticent target sequence of using.

WO2005/047505 relates to Microrna, produces the method for Microrna and the method for using Microrna.

WO 2005/078096 part is based on following discovery: interior miRNAs can be raised the translation repression said target mrna.Reticent agent of the described RNA of this article and method provide by inhibition for example synthesizes the means for the treatment of heredity (for example, heredity neurodegenerative disease, for example, Huntington chorea) or nongenetic disease in disease proteinic.Therefore, the reticent agent of described RNA has mRNA targeting moiety, connection portion and miRNA and raises part.

WO2005/100574 relates to the means and the method for the biomass yield of improveing plant and/or plant-growth and/or plant structure.Especially, its concern illustrates the biomass yield of increase and the transgenic plant of plant growth rate than corresponding wild type plant show.These plant characteristics are to contain the Microrna that level changes, particularly the member's of the SPL gene family of target coding SPL transcription factor Microrna.

WO2006/034368 has described miRNA, particularly from the miRNA of willow, is used for plant-growth and growth.

WO2006/044322 has described composition and the method that can be used for target sequence containment, target sequence affirmation and target sequence downward modulation.This article provides the polynucleotide constructs that can be used for gene silencing or RNA downward modulation, and the cell, the Plants and Seeds that comprise described polynucleotide.It also provides the method for using reticent target sequence of Microrna or downward modulation RNA.

People such as Abreu (2004, Genes and Development 18:2237-2242) have described in the body to the functional dependency of the transcribing of Mirnas of plant, processing, inscribe lytic activity and spatial distribution and have studied.For reaching this purpose, made up synthetic miRNA---the miR171 of target GFP coding region.

(2005, Developmental Cell 8:517-527) has described the specific function of miRNA for plant transcription thing group to people such as Schwab, and has released one group of empirical parameter and be used for target identification, and this can be applicable to synthetic miRNA.

People such as Schwab (2006, Plant Cell, 18 (5): 1121-1133) described the high specific gene silencing that is undertaken by artificial mi RNA in Arabidopis thaliana (Arabidopsis), described miRNA is by target, to reduce heterogeneic expression.

People such as Alvarez 2006 (Plant Cell.2006 May; 18 (5): 1134-1151) describe: endogenous and synthetic Microrna can be when different plant species (comprising tobacco and tomato) moderate stimulation be to multiple target, the regulation and control of efficient and localization.

WO 00/04173 has described the method for regulating programmed cell death (PCD) in eukaryotic cell and biology (particularly vegetable cell and plant), this realizes that by introducing " PCD regulates mosaic gene " described " PCD regulates mosaic gene " can influence endogenous poly-(ADP-ribose) polysaccharase (PARP) expression of gene and/or apparent activity.Programmed cell death can be suppressed or excite.This invention is particularly related to coding to have the active proteinic nucleotide sequence of PARP and is used to regulate PCD, is used to strengthen growth velocity or is used to produce the stress-tolerance sexual cell and biological purposes.

WO 2004/090140 has described by reducing in the plant endogenous PARG activity of proteins increases that plant is coerced for inanimate or the ways and means of the tolerance of unfavorable growth conditions (comprising arid, highlight strength, high temperature, nutrition restriction etc.).

WO 2006/045633 has described the purposes that cotton parp2 gene or cDNA sequence are used to obtain the stress tolerance vegetable lamb.Multiple cotton parp2 sequence also is provided.

Above-mentioned prior art document all relates to the miRNA that reply of regulation and control plant to disadvantageous inanimate condition less than describing, and does not also describe the purposes that it is used to improve stress tolerance in plants.Do not had description of the Prior Art yet and can be used for improveing the synthetic miRNA of stress tolerance in plants.

The purpose of this invention is to provide this type of and coerce relevant miRNA molecule and synthetic miRNA molecule, they can be used for improveing or increase the tolerance of plant for unfavorable growth conditions, and make them have more resistance for stress conditions (for example inanimate stress conditions).

Summary of the invention

In one embodiment, the invention provides separated RNA molecule or dna molecular, it comprises arbitrary nucleotide sequence among SEQ ID NO:1 to the SEQ ID NO:36 or its can instruct variant to the cutting of identical target sequence.

In another embodiment, the invention provides mosaic gene, described mosaic gene comprise can be in plant expression promoter, described promotor effectively links to each other with the DNA zone, described DNA zone is processed into miRNA at the introduced plant cell and after transcribing therein, and described DNA zone comprises arbitrary nucleotide sequence among SEQ ID NO:1 to the SEQ ID NO:36 or its can instruct variant to the cutting of identical target sequence; And, randomly, relate to 3 ' DNA zone of Transcription Termination and poly-adenosineization.Can in plant, expression promoter can be constitutive promoter or tissue-specific promoter or inducible promoters.

In another embodiment, the invention provides the vegetable cell or the plant that comprise following mosaic gene, described mosaic gene comprise can be in plant expression promoter, described promotor effectively links to each other with the DNA zone, described DNA zone is processed into miRNA at the introduced plant cell and after transcribing therein, and described DNA zone comprises arbitrary nucleotide sequence among SEQ ID NO:1 to the SEQ ID NO:36 or its can instruct variant to the cutting of identical target sequence; And, randomly, relate to 3 ' DNA zone of Transcription Termination and poly-adenosineization.

The present invention also provides and has been used to increase the method for plant for the resistance of unfavorable growth conditions, described method comprises the steps: in the cell of described plant to introduce following mosaic gene, and described mosaic gene comprises the following DNA zone that effectively links to each other: can be in plant expression promoter; The DNA zone, described DNA zone is processed into miRNA at the introduced plant cell and after transcribing therein, and described miRNA can discern the cutting that and guides the mRNA of the endogenous PARP gene of plant or ParG gene; And, randomly, relate to 3 ' DNA zone of Transcription Termination and poly-adenosineization.

Preferably, the DNA zone of the coding miRNA that mentions comprises the basic complementary nucleotide sequence of nucleotide sequence with at least 21 continuous nucleotides of the endogenous PARP gene of plant or ParG gene, and condition is to allow the mispairing between the corresponding nucleotide sequence in terminal Nucleotide of one or more following mispairing: miRNA 5 ' and the RNA molecule; Mispairing between the corresponding nucleotide sequence in the arbitrary and RNA molecule in the Nucleotide on the 1st to the 9th of miRNA; Three mispairing between the corresponding nucleotide sequence in the arbitrary and RNA molecule in the Nucleotide on the 12nd to the 21st of miRNA, prerequisite is to be no more than two continuous mispairing.Mosaic gene can comprise the nucleotide sequence of 709 Nucleotide of the 689th Nucleotide to the of SEQ ID NO:37.Mosaic gene also can comprise the nucleotide sequence of SEQ ID NO:38, SEQ ID NO:40, SEQ ID NO:41 or SEQ ID NO:42.Mosaic gene codified according to the present invention can be processed into the primary transcribe of preceding-miRNA, wherein, described before-miRNA be derived from naturally occurring before-miRNA, for example-miR398, and wherein, described primary transcribe can adopt secondary RNA structure, it comprises single stranded RNA structure and double-stranded RNA stem and preceding-miRNA processing signal, before making-single stranded RNA structure in miRNA processing signal zone and the binding (junction) between the double-stranded RNA stem similar naturally occurring before-miRNA or pri-mRNA in before-miRNA processing signal zone in binding between single stranded RNA structure and the double-stranded RNA stem.

Mosaic gene therefore can comprise naturally occurring before-miRNA, for example before-nucleotide sequence of miR398 (that is, do not have naturally occurring miRNA and miRNA as support ^*Sequence), for example, the nucleotide sequence of SEQID NO:41, and the nucleotide sequence of 147 Nucleotide of the 119th Nucleotide to the of SEQ ID NO:41 from 97 Nucleotide of the 55th Nucleotide to the of the nucleotide sequence of 33 Nucleotide of the 1st Nucleotide to the and SEQ IDNO:41.

In another embodiment of the invention, the vegetable cell and the plant that following mosaic gene are provided and have comprised this type of mosaic gene, described mosaic gene comprises the following DNA zone that effectively links to each other: can be in plant expression promoter; The DNA zone, described DNA zone is processed into miRNA at the introduced plant cell and after transcribing therein, and described miRNA can discern the cutting that and guides the mRNA of the endogenous PARP gene of plant or ParG gene; And, randomly, relate to 3 ' DNA zone of Transcription Termination and poly-adenosineization.

The present invention also provides and can discern the synthetic miRNA that and guides the cutting of the mRNA of the endogenous PARP gene of plant or ParG gene, preferably, such miRNA, it comprises the basic complementary nucleotide sequence of nucleotide sequence with at least 21 continuous nucleotides of the endogenous PARP gene of plant or ParG gene, and condition is to allow the mispairing between the corresponding nucleotide sequence in terminal Nucleotide of one or more following mispairing: miRNA5 ' and the RNA molecule; Mispairing between the corresponding nucleotide sequence in the arbitrary and RNA molecule in the Nucleotide on the 1st to the 9th of miRNA; Three mispairing between the corresponding nucleotide sequence in the arbitrary and RNA molecule in the Nucleotide on the 12nd to the 21st of miRNA, prerequisite is to be no more than two continuous mispairing.

In another embodiment of the present invention, provide and be used for reducing the method for miRNA for the influence that reduces the vegetable cell expression of target gene, described method comprises the steps: to provide the excessive RNA substrate of being discerned and cutting by miRNA to vegetable cell, and this RNA substrate is discerned and cut by described miRNA.

According to preamble and other target, advantages and features of the invention will be apparent hereinafter, can more be expressly understood essence of the present invention with reference to hereinafter to detailed description, claims and the accompanying drawing of the different embodiments of the present invention.

The accompanying drawing summary

The synoptic diagram that Fig. 1: miRNA 171 supports change is to produce the miRNA of the endogenous PARP gene of target.The nucleotide sequence of figure A:mRNA 171.Figure B: the miRNA 171 after the miR171 sequence of the PARP sequence replacement 21nt of usefulness 21nt.Figure C: by to miRNA ^*Introduce the mRPARP sequence that two place's projectioies (bulge) obtain in the sequence.Δ G is Gibb ' the s free energy of the optimum RNA structure that folds.The end points of arrow indication miRNA sequence.

Fig. 2: to the comparison of sequence identity degree between the corresponding sequence in miRPARP, miRPARP2m and miRPARP3m and the target gene (Parp 1 or Parp 2).MiRPARP2m has the mispairing of two places, and miRPARPm3 has the mispairing of three places.

Fig. 3: to the design of the miRNA that can discern Arabidopis thaliana PARP1 and PARP2 gene.Mispairing between miRNA and target nucleotide sequences illustrates with more shallow color.

Fig. 4: to the modification on preceding miRNA398a (left side), with the nucleotide sequence (right side) that mixes miRPARP7.Microrna illustrates with runic, and the complementary strand of Microrna illustrates with italic.

Fig. 5: to can specific recognition the design of miRNA of Arabidopis thaliana PARP2 gene.Mispairing between miRNA and target nucleotide sequences illustrates with more shallow color.

Fig. 6: to the modification on preceding miRNA398a (left side), with the nucleotide sequence (right side) that mixes miRPARP2-8.Microrna illustrates with runic, and the complementary strand of Microrna illustrates with italic.

Fig. 7: the Northern blot hybridization, with identify express produce before-Microrna through processing of 21 Nucleotide in the transgenic arabidopsis of the mosaic gene of Microrna 2-8.Left figure (A): with the oligonucleotide probe hybridization of specific recognition miRPARP2-8; Right figure (B): use specific recognition miRPARP2-8 ^*The oligonucleotide probe hybridization of chain.

Fig. 8: RT-PCR is quantitative, to detect the PARP2 mRNA accumulation in the transgenic arabidopsis of expressing preceding-Microrna 2-8 mosaic gene.PTMBO024-1 is different transgenic lines with pTMBO024-3, and pTMBO009 comprises uncorrelated genetically modified transgenic lines.HL represents: growing under high optical condition before the separating mRNA.

Fig. 9: figure A: before the prediction secondary RNA structure of not modified primary transcribe (pri-miR171), described primary transcribe are processed into-miR171 (1), finally be processed into miR171.Figure B: before being processed into-miRPARP (2), finally be processed into the prediction secondary RNA structure of the transcript of miRPARP.Figure C: the frame inner structure of figure A is processed the amplification that site (10) is located at preceding-miR171.(3): length is the double-stranded RNA stem of about 8 Nucleotide.(4) and the non-paired rna structure of (12) strand; (5) through processing the end points of miRNA171.Figure D: the frame inner structure of figure B is processed the amplification that site (11) is located at preceding-miRPARP.(6): the NcoI cloning site; (7) NheI cloning site; (8) length is the double-stranded RNA stem of about 12 Nucleotide; (9) and (13): the non-paired rna structure of strand; (14): through the prediction end points of processing miRNAPARP.

Figure 10: figure A: before the prediction secondary RNA structure of not modified primary transcribe (pri-miR398), described primary transcribe are processed into-miR398 (1), finally be processed into miR398.Figure B: before being processed into-miRPARP2-8 (2), finally be processed into the prediction secondary RNA structure of the transcript of miRPARP2-8.Figure C: the mount structure of figure A is processed the amplification that site (11) is located at preceding-miR398.(4): length is the double-stranded RNA stem of about 7 Nucleotide.(5) and the non-paired rna structure of (13) strand; (3) through processing the end points of miRNA398.Figure D: the mount structure of figure B is processed the amplification that site (12) is located at preceding-miRPARP2-8; (6): the non-paired rna structure of strand (7); (8) non-structure (unstructured) the RNA zone of about 7 Nucleotide; (9) through processing the prediction end points of miRPARP; (10): NheI and NcoI cloning site are positioned at the back of single stranded RNA projection.

The detailed description of different embodiments

The present invention is based on evaluation to the miRNA molecule of novelty, described molecule can reduce following endogenous plant expression of gene, described endogenous plant gene has been presented at and has been subjected to the otherness regulation and control in experience plant of different inanimate stress response or the vegetable cell, perhaps has been presented at according to W000/004173 or the described method of WO2004/090140 and is subjected to the otherness regulation and control in transforming the plant that has more stress tolerance or vegetable cell as.Another aspect of the present invention is based on following situation: surprisingly, the introducing of the mosaic gene of coding synthetic miRNA molecule (the endogenous PARP gene as target sequence is discerned in the plant in design) enough realizes the downward modulation to the described endogenous PARP gene of W000/004173 effectively.

Therefore, in the first embodiment, the invention provides and relate to the miRNA molecule of regulating and control following gene expression in plants, described plant gene relates to vegetable cell to the replying of stress conditions (particularly inanimate stress conditions), and the present invention also provides this type of miRNA molecule to be used to obtain the purposes of stress tolerant plants cell and plant.This type of miRNA comprises following synthetic miRNA molecule, and they are instructed with cutting to transcribe next mRNA molecule from the endogenous PARP or the PARG encoding gene of vegetable cell.

When using in this article, " miRNA " is that length is the RNA molecule of about 20 to 22 Nucleotide, it can be loaded onto in the RISC complex body, and instruct cutting to another RNA molecule, wherein, other RNA molecule comprises and the basic complementary nucleotide sequence of the nucleotide sequence of miRNA molecule, wherein, one or more following mispairing can take place:

Mispairing in the Nucleotide of the 5 ' end of described miRNA and the target RNA molecule between the corresponding nucleotide sequence;

Mispairing between the corresponding nucleotide sequence in the arbitrary and target RNA molecule in the Nucleotide on the 1st to the 9th of described miRNA;

Three mispairing between the corresponding nucleotide sequence in the arbitrary and target RNA molecule in the Nucleotide on the 12nd to the 21st of described miRNA, prerequisite is to be no more than two continuous mispairing.

On the 10th and 11 of miRNA, do not allow mispairing (all miRNA positions all are that 5 ' end from the miRNA molecule begins to represent).

MiRNA by " preceding-miRNA " molecule by the protein that exists in any vegetable cell for example the processing of DCL protein obtain, and be loaded into the RISC complex body, wherein it can instruct the cutting to target RNA molecule.

Preceding-microRNA molecules normally obtains from pri-microRNA molecules (primary transcribe) processing.In animal, the maturation of Microrna by the Drosha-DGCR8 complex body by coming initial to the clean cut that is embedded in the stem ring in the primary transcribe.Before-the single stranded RNA segment of Microrna flank for before pri-miRNA is processed as-miRNA is important.Cleavage site look like by with stem-ssRNA banded distance decide (people 2006 such as Han, Cell 125,887-901,887-901).

When using in this article, " preceding-miRNA " molecule is the about 100 RNA molecules to about 200 Nucleotide, preferably approximately 100 is to about 130 Nucleotide, it can adopt following secondary structure, described secondary structure comprises double-stranded RNA stem and single stranded RNA ring, and the nucleotide sequence (and complementary sequence) that also comprises miRNA in the double-stranded RNA stem.Preferably, miRNA and complement thereof are positioned at apart from miRNA double-stranded RNA stem free-end about 10 to about 20 Nucleotide places.The length in single-stranded loop zone and sequence are not crucial, and it can change to a great extent, for example, in length between 30 to 50nt.Before the synthetic-example of miRNA is shown in Fig. 1.Some other example is shown in Fig. 4 and 6.Preferably, non-in pairs and the free energy difference between the paired rna structure-20 between-60kcal/ the mole, especially, about-40kcal/ mole.MiRNA and miRNA ^*Between complementation need not be complete, the projection of about 1 to 3 non-paired Nucleotide in place is an acceptable.Can be by traditional computerized algorithm in this area, for example mFOLD predicts the secondary structure that the RNA molecule adopts.By the active specific chains of double-stranded RNA stem that discharges and be loaded into the preceding-miRNA on the RISC complex body of DCL by 5 ' terminal complementary degree decision, wherein, hydrogen-bonded chain between the Nucleotide of its 5 ' terminal different chains that relate to the dsRNA stem that is cut at least is loaded onto on the RISC complex body, and it will determine the sequence-specific of target RNA molecular degradation.But, rule of thumb, if the miRNA molecule from specific synthetic preceding-miRNA molecule does not have function (because having loaded " mistake " chain on the RISC complex body), this problem can bring solution by the position of miRNA molecule and its complement on each chain of the dsRNA stem of preceding-miRNA molecule are intersected so, and this will be conspicuous at once.As known in the art, it is strong not as the combination that relates to three hydrogen bonds between G and the C to relate to the combination that relates to two hydrogen bonds between two hydrogen bonds or G and the U between A and the U.

Naturally occurring miRNA molecule can be comprised in their naturally occurring preceding-miRNA intramolecularly, but they also can be introduced into existing before-miRNA divides submounts, this by before will be from this type of existing-nucleotide sequence of the miRNA molecule that miRNA molecule normal process obtains and the nucleotide sequence of another miRNA interested exchange and realize.The support of preceding-miRNA also can be complete synthetic.Similarly, synthetic miRNA molecule can be comprised in existing before-before miRNA divides submounts or synthetic-the miRNA support in, or get from its processing.Interesting ground, observe, before some-miRNA supports correctly are processed as at it is better than other aspect efficient of Microrna of design, when particularly expressing as mosaic gene, in described mosaic gene, except preceding-Microrna, other DNA zone, for example, untranslated leader or Transcription Termination and poly-adenosine zone also are merged in primary transcribe.Especially, have been found that comprise before-Microrna and other be before more or less the transcript in complementary zone can disturb primary transcribe correctly to be processed as-Microrna and the Microrna that finally is processed as design.Before other-and the Microrna support, for example preceding Microrna 398 may be difficult for being carried out this type of incorrect processing.Do not desiring to limit the invention under the situation of specific function pattern, estimate, in most of on the whole double-stranded RNA zone, locate the existence in one or more non-structure strands zone apart from the specific fixed position of preceding-miRNA molecule cleavage site, with the correct processing that has influence on the preceding-miRNA molecule of design, and act on the following fact, for example, for from before being derived from of primary transcribe-the support processing of miR398 than for example to from before being derived from of primary transcribe-the more difficult generation mistake of processing of the support of miR171.

Apparent for those skilled in the art: the existence of additional sequences may particularly impact the projection in other double-stranded RNA stem (Asia) structure or the existence and the position of single stranded RNA structure for folding the impacting of primary transcribe RNA molecule to secondary RNA structure.Single stranded RNA or bulge-structure are with respect to the position of preceding-miRNA, that is, the distance of Nucleotide should carefully be kept.For specific RNA nucleotide sequence, can use Software tool well known in the art and algorithm, for example (people 2003 Nucleic Acids Research 31 such as Zucker 3406-3415) easily predict secondary RNA structure to MFOLD.In addition, known in this field, can design or modified nucleotide by substituted nucleotide in nucleotide sequence, the feasible new Nucleotide of introducing shows another part complementarity more or less with nucleotide sequence, and influences the generation of double-stranded RNA stem or single stranded RNA projection by this way.

In one embodiment of the invention, novel miRNA molecule is provided, they relate to being coerced plant and being coerced the regulation and control of the plant gene of being regulated and control by difference between plant, wherein, described miRNA molecule comprises arbitrary nucleotide sequence among SEQ ID NO:1 to the SEQ ID NO:36.The present invention also provides its variant, and described variant can instruct the cutting according to identical with the miRNA molecule of mentioning at least target RNA sequence of above-mentioned mispairing rule, and preceding-miRNA molecule and variant thereof that the nucleotide sequence that comprises the miRNA molecule is provided.Preceding-the miRNA molecule (and also having the miRNA molecule thus) be the introduced plant cell easily, this realizes by following gene is provided to vegetable cell, described gene comprise can be in plant expression promoter, before described promotor produces when transcribing-the DNA zone of miRNA molecule links to each other effectively.Can in plant expression promoter can be and the natural relevant promotor of preceding-miRNA molecule, perhaps can be allogeneic promoter.

Novel miRNA molecule, the preceding-miRNA that perhaps is processed as this type of miRNA molecule can be used for modifying the regulation and control to target gene, and improvement vegetable cell replying unfavorable growth conditions thus.In a kind of mode, the miRNA molecule that exists in the described vegetable cell can become functional less or do not have function, causes being subjected under the normal circumstances target gene expression of novel miRNA regulation and control to increase.It is functional less or do not have function to realize in many ways that miRNA is become, and comprises the selection of carrying out at the variation in the nucleotide sequence of miRNA encoding sequence (being included in T-DNA inserts after the mutagenesis or the selection of carrying out after inducing mutagenesis).Make that another less approach of miRNA molecular function is the amount that increases target RNA molecule (perhaps being its part of being discerned by miRNA at least).This increase of target RNA can realize easily by following mosaic gene is provided to vegetable cell, described mosaic gene comprise can be in plant the DNA zone of expression promoter and encode this type of target RNA or its part.In another embodiment, the DNA of coding miRNA resistance target RNA also can link to each other with the relevant promotor of preceding-miRNA natural and the identification target gene, and is introduced into vegetable cell.By this way, miRNA resistance target RNA will express under the situation identical with miRNA, and resistance target RNA will replace the non-resistance target RNA by miRNA inductive cutting degraded.MiRNA resistance target RNA and modified make its to the miRNA inductive be cut with resistance (for example, by modify its sequence make to the 10th or 11 the Nucleotide complementary target sequence Nucleotide of miRNA in introduce to change cause mispairing to realize) target RNA similar substantially.Clearly, if target RNA is the RNA of coded protein, it is reticent that so any modification all needs coding region, perhaps causes producing the replacement of functional protein at least.Non-functional miRNA allelotrope or miRNA resistance target gene also can be introduced by homologous recombination, to replace miRNA coding allelotrope or miRNA susceptibility target gene.

The silence of target gene or the increase of expression of target gene reduction also can be realized by the level that increases miRNA in the vegetable cell.Easily, this can realize that described mosaic gene comprises the following dna fragmentation that effectively links to each other by following mosaic gene is provided to vegetable cell:

Can be in plant expression promoter,

The DNA zone, it is processed into miRNA or its variant according to novelty of the present invention after being introduced into vegetable cell and transcribing in vegetable cell; And

Randomly, 3 ' the DNA zone that relates to Transcription Termination and poly-adenosineization.

In another embodiment of the present invention, providing increases the method for plant for the resistance of unfavorable growth conditions, wherein, vegetable cell with synthetic miRNA molecule is provided, and the cutting of the mRNA that endogenous PARP gene or PARG genetic transcription from plant are come is discerned and instructed to described synthetic miRNA molecular energy.Preferably, this type of miRNA molecule comprise with PARP or PARG gene (preferably, endogenous PARP gene of plant or ParG gene (comprise hereinafter described plant PARP or any sequence in the PARG sequence)) the basic complementary nucleotide sequence of nucleotide sequence of at least 21 continuous nucleotides, condition is to allow one or more following mispairing:

Mispairing in the Nucleotide of the 5 ' end of described miRNA and the RNA molecule between the corresponding nucleotide sequence;

Mispairing between the corresponding nucleotide sequence in the arbitrary and RNA molecule in the Nucleotide on the 1st to the 9th of described miRNA;

Three mispairing between the corresponding nucleotide sequence in the arbitrary and RNA molecule in the Nucleotide on the 12nd to the 21st of described miRNA, prerequisite is to be no more than two continuous mispairing.

On the 10th and 11 of miRNA, do not allow mispairing.

This type of miRNA can with before following-form of miRNA offers plant easily, described before-miRNA comprises the nucleotide sequence of miRNA in its double-stranded RNA stem, as described in other place of the application.Before-the miRNA support can from before existing-miRNA, perhaps can be before synthetic, the synthetical-the miRNA support.Most convenient ground, before coding comprises this type of of miRNA-the DNA zone of miRNA with can be in plant expression promoter and the optional 3 ' end that relates to Transcription Termination and poly-adenosineization link to each other effectively, and be introduced into vegetable cell in mode well known in the art (comprise conversion and hybridize).

When using in this article, " stress tolerant plants " or " to the plant of stress conditions or the tolerance of unfavorable growth conditions " is such plant (the particularly plant of the method according to this invention acquisition), it grows better than the control plant of not handling according to method of the present invention when the unfavorable growth conditions of experience (such as but not limited to arid, high temperature, limited, the highlight strength of nutrition (particularly nitrogen) supply) certain hour.This is just apparent from the general outward appearance of plant usually, and can be for example by increasing biomass production, under unfavourable condition, continue nourish and grow or higher seed production is measured.Stress tolerant plants has wider auxanogram, that is, weather and other inanimate that they can stand wider scope change, and do not have output punishment.From biochemical angle, stress tolerance can obviously be shown as: under the stress conditions, stress tolerant plants is than control plant and the NAD of Yan Genggao ⁺-NADH/ATP content and lower active oxygen classification produce.Stress tolerance also can obviously be shown as: under the stress conditions, and the chlorophyll content of stress tolerant plants control plant and Yan Genggao under the same terms, higher photosynthesis and lower chlorophyll fluorescence.

Should be understood that, obviously manifest, need not under unfavourable condition, grow continuously by plant in order to make stress tolerance.Usually, even only run into the unfavourable condition of relative short duration at growing period, just will be obviously according to the difference of the stress tolerance between plant of the present invention or vegetable cell and control plant or vegetable cell.

Can identify following data base entries, it has identified poly polymerase protein sequence experiment shows and supposition, its part or homologous sequence: BAD53855 (rice (Oryza sativa)), BAD52929 (rice), XP_477671 (rice), BAC84104 (rice), AAT25850 (Zea mays (Zea mays)), AAT25849 (Zea mays), NP_197639 (mouse ear mustard (Arabidopsisthaliana)), NP_850165 (mouse ear mustard), NP_188107 (mouse ear mustard), NP_850586 (mouse ear mustard), BAB09119 (mouse ear mustard), AAD20677 (mouse ear mustard), Q11207 (mouse ear mustard), C84719 (mouse ear mustard), T51353 (mouse ear mustard), T01311 (mouse ear mustard), AAN12901 (mouse ear mustard), AAM13882 (mouse ear mustard), CAB80732 (mouse ear mustard), CAA10482 (mouse ear mustard), AAC79704 (Zea mays), AAC19283 (mouse ear mustard), CAA10888 (Zea mays), CAA10889 (Zea mays), CAA88288 (mouse ear mustard).

With regard to purpose of the present invention, the protein of PARP protein is defined as having poly (ADP-ribose) polymerase activity preferably, comprises so-called " PARP label ".The PARP label is the aminoacid sequence of high conservative between PARP protein, and it is defined as from proteinic the 858th amino acids of house mouse (Mus musculus) PARP by de Murcia and Menussier deMurcia (1994) and extends to the 906th amino acids.This structural domain is corresponding to zeistic conventional PARP protein (ZAP1; The SEQ ID NO:2 of WO 00/04173, the document is incorporated this paper by reference into) the 817th to 865 amino acids sequence, perhaps corresponding to zeistic conventional PARP protein (ZAP2; The SEQ ID NO:11 of WO 00/04173, the document is incorporated this paper by reference into) the 827th to 875 amino acids sequence, perhaps corresponding to zeistic unconventional PARP protein (the SEQ ID NO:4 of WO 00/04173, the document is incorporated this paper by reference into) the 500th to 547 amino acids sequence, perhaps corresponding to the 485th to 532 amino acids sequence of the unconventional PARP protein (the SEQ ID NO:6 of WO00/04173, the document is incorporated this paper by reference into) of mouse ear mustard.This aminoacid sequence is high conservative (having about 90% to 100% sequence identity) between different PARP protein.Conservative especially is from the Methionin on proteinic the 891st of the PARP of house mouse (corresponding to the 850th of the SEQID NO:2 of the WO 00/04173 that incorporates this paper by reference into, the 861st of SEQ ID NO:11 who incorporates the WO 00/04173 of this paper by reference into, the 532nd of SEQ ID NO:4 who incorporates the WO 00/04173 of this paper by reference into, the 517th of SEQ ID NO:6 who incorporates the WO 00/04173 of this paper by reference into), it is considered to relate to the proteinic catalytic activity of PARP.Especially, the PARP of house mouse is proteinic the 865th, 866,893,898 and 899 or be variable for the amino acid on the corresponding position of other sequence.PARP albumen also can comprise terminal DNA binding domains of N-and/or nuclear localization signal (NLS).

At present, two class PARP protein have been described.As defined herein, the first kind comprises so-called classics and contains Zn and refer to PARP protein (ZAP).These protein sizes are characterised in that in the scope of 113-120kDA in addition: be arranged at least one of protein N-end structure territory (particularly be positioned at protein first amino acid whose about 355 to about 375), preferred two Zn finger domains exist.Zn refers to be defined as to have the peptide sequence of sequence C xxCxnHxxC (wherein n can 26 to 30 variations) that can compound Zn atom.Comprising from the example of the proteinic aminoacid sequence of PARP of ZAP class can be with accession number P18493 (ox (BosTaurus)) in the PIR albumen database, P26466 (jungle fowl (Gallus gallus)), P35875 (drosophila melanogaster (Drosophilamelanogaster)), P09874 (people (Homo sapiens)), P11103 (house mouse), Q08824 (Oncorynchus masou), P27008 (Rattus norvegicus (Rattus norvegicus)), Q11208 (sarcophaga fuscicauda (Sarcophaga peregrine)), the sequence that P31669 (smooth Xenopus laevis (Xenopus laevis)) finds and identify at present from zeistic ZAP1 and the proteinic sequence of ZAP2 (the SEQ ID NO:2/ that incorporates the WO 00/04173 of this paper by reference into incorporates the SEQ ID NO:11 of the WO 00/04173 of this paper by reference into).

The nucleotide sequence of corresponding cDNA can find in the EMBL database and at SEQ ID NO:1 and 10 (Zea mays) with accession number D90073 (ox), X52690 (jungle fowl), D13806 (drosophila melanogaster), M32721 (people), X14206 (house mouse), D13809 (Oncorynchus masou), X65496 (Rattus norvegicus), D16482 (Sarcophagaperegrina), D14667 (smooth Xenopus laevis).

As herein defined, second class comprises so-called non-classical PARP protein (NAP).These protein less (72-73kDa), and be characterised in that at proteinic N-end there is not the Zn finger domain in addition, and comprise the existence in N-end structure territory that has the amino acid section of similarity with dna binding protein dna.Preferably, this class PARP protein comprises about 30 to 32 amino acid whose at least one aminoacid sequences, and (amino acid illustrates with standard single-letter code, and wherein x represents any amino acid wherein to comprise sequence R G x x x x G x K x x x x x R L; The SEQ ID NO:7 that incorporates the WO 00/04173 of this paper by reference into).Even more preferably, these PARP protein comprise about 32 amino acid whose following at least one aminoacid sequences, it has sequence x L x Vx x x R x x L x x R G L x x x G V K x x L V x R L x x A I (the SEQ ID NO:8 that incorporates the WO 00/04173 of this paper by reference into) (so-called A1 structural domain), perhaps comprise about 32 amino acid whose following at least one aminoacid sequences, it has sequence G M x x x E L xx x A x x R G x x x x G x K K D x xR L x x (the SEQ ID NO:9 that incorporates the WO00/04173 of this paper by reference into) (so-called A2 structural domain), or the both comprises.Especially, A1 and A2 structural domain can form the helix-loop-helix structure.These PARP protein also can comprise alkalescence " B " structural domain (about 35 aminoacid sequences to about 56 amino acid whose K/R of being rich in relate to the protein target to nuclear) and/or acid " C " structural domain (aminoacid sequences of about 36 amino acid whose D/E of being rich in).From the example of the protein sequence of NAP class comprise from mouse ear mustard app protein (can obtain with accession number Q11207 from the PIR Protein Data Bank, incorporate the SEQ ID NO:6 of the WO 00/04173 of this paper by reference into) and from zeistic NAP protein (SEQ ID NO:4).The sequence of corresponding cDNA can find in the EMBL database and in the SEQ ID of the WO 00/04173 that incorporates this paper by reference into NO:3 with accession number Z48243 (the SEQ ID NO:5 that incorporates the WO 00/04173 of this paper by reference into).

Clearly, with regard to purpose of the present invention, can be from other plant species or kind, particularly separate or identify coding from the PARP protein of defined above-mentioned two classes or other gene or the cDNA of its part from other plant or kind.Can for example pass through Southern blot hybridization (low preciseness or the hybridization of high preciseness, depend on the species of thinking therefrom to separate the PARP gene and probe ultimate source from species between relation) separate these PARP gene or cDNA, wherein use dna fragmentation as probe, described fragment has nucleotide sequence or its part of PARP gene mentioned above or cDNA, preferably, conservative part for example, comprises the gene fragment of the nucleotide sequence of the PARP label mentioned above of encoding.Corresponding to the nucleotide sequence of the proteinic PARP label of PARP of plant gene coding is the nucleotide sequence of the 2558th to 2704 Nucleotide of SEQ ID NO:1 of incorporating the WO 00/04173 of this paper by reference into, or incorporate the nucleotide sequence of the 1595th to 1747 Nucleotide of SEQ ID NO:3 of the WO00/04173 of this paper by reference into, perhaps incorporate the nucleotide sequence of the 1575th to 1724 Nucleotide of SEQ ID NO:5 of the WO 00/04173 of this paper by reference into.If between the class of PARP gene, distinguish, should preferably use the special part for class of PARP gene so, for example, the N-end structure territory before catalyst structure domain or its part.

Alternatively, also can separate the gene or the cDNA of coding PARP protein or its part by pcr amplification, wherein use suitable primer, for example, have SEQ ID NO:13, incorporate the degenerated primer of the nucleotide sequence of the sequence shown in the SEQ ID NO:14 of WO 00/04173 of this paper into by reference, perhaps have primer corresponding to the nucleotide sequence of the sequence shown in the SEQ ID NO:15 to 20 of the WO 00/04173 that incorporates this paper by reference into corresponding to the WO 00/04173 that incorporates this paper by reference into.But, clearly, those skilled in the art can design alternative oligonucleotide and be used for PCR, perhaps can use at least 20 that comprise any PARP gene by pcr amplification, preferably about at least 30, the oligonucleotide of the nucleotide sequence of special about at least 50 continuous nucleotides.

Clearly, obtainable to those skilled in the art, be used for coming these technology of isolated genes or cDNA or other technology (to comprise based on part fragment and nucleotide sequence thereof (for example obtaining) by pcr amplification, RACE-PCR for example) combination can be used for separating the PARP gene or its part that are applicable to method of the present invention.

In addition, PARP gene (coding some of them amino acid has been replaced the PARP protein of (so-called conservative replacement) by some other chemically similar amino acid) or synthetic PARP gene (its coding protein similar but be based on the genetic code degeneracy have the different IPs nucleotide sequence) and partly also be applicable to method of the present invention to natural PARP gene.

The nucleotide sequence of parp2 gene or parp2 cDNA can be included in the nucleotide sequence of any one parp2 variant of identifying in the cotton variety, comprise: the SEQ ID NO:5 that incorporates the WO2006/045633 of this paper by reference into, the SEQ ID NO:6 that incorporates the WO2006/045633 of this paper by reference into, the SEQ ID NO:7 that incorporates the WO2006/045633 of this paper by reference into, the SEQ ID NO:8 that incorporates the WO2006/045633 of this paper by reference into, the SEQ ID NO:9 that incorporates the WO2006/045633 of this paper by reference into, the SEQ ID NO:10 that incorporates the WO2006/045633 of this paper by reference into, the SEQ ID NO:11 that incorporates the WO2006/045633 of this paper by reference into, the SEQ ID NO:12 that incorporates the WO2006/045633 of this paper by reference into, the SEQ ID NO:18 that incorporates the WO2006/045633 of this paper by reference into, incorporate into by reference this paper WO2006/045633 SEQ ID NO:19 or incorporate the nucleotide sequence of SEQ ID NO:20 of the WO2006/045633 of this paper by reference into, or coding comprises the SEQ ID NO:13 of the WO2006/045633 that incorporates this paper by reference into, incorporate into by reference this paper WO2006/045633 SEQ ID NO:21 or incorporate proteinic nucleotide sequence or its variant of aminoacid sequence of SEQ ID NO:22 of the WO2006/045633 of this paper by reference into.

When using in this article, " PARG encoding gene " or " ParG gene " is the proteinic gene of PARG (poly ADP ribose glycogen lytic enzyme) of encoding, wherein, the depolymerization of PARG protein catalysis poly ADP ribose, this realizes by discharging free ADP ribose by inscribe glycolysis-(endoglycolytic) or circumscribed glycolysis-(exoglycolytic) effect.

The PARG encoding gene can comprise the nucleotide sequence of the following proteins of encoding, described protein comprises the SEQ ID NO:1 (mouse ear mustard) of the WO 2004/090140 that incorporates this paper by reference into or incorporates the SEQ ID NO:2 (potato (Solanumtuberosum)) of the WO 2004/090140 of this paper or aminoacid sequence or its part of SEQ ID NO:16 (rice) by reference into, the for example following dna fragmentation of described gene, it comprises the SEQID NO:3 of the WO 2004/090140 that incorporates this paper by reference into, incorporate into by reference this paper WO 2004/090140 SEQ ID NO:4 or incorporate the SEQ ID NO:15 of WO 2004/090140 of this paper or the nucleotide sequence of SEQ ID NO:23 (Zea mays) by reference into.

But should be understood that, technician's useful source from from the probe of the above-mentioned PARG encoding gene of plant species or even use the probe that is derived from from the above-mentioned PARG encoding gene of animal species, separate modification D NA sequence by hybridization from other plant species.For reaching this purpose, probe should preferably have following nucleotide sequence, and described nucleotide sequence comprises at least 40 continuous nucleotides from the coding region of PARG coding gene sequence mentioned above (preferably from the SEQ ID NO:3 of the WO 2004/090140 that incorporates this paper by reference into or from the coding region of the SEQ IDNO:4 of the WO 2004/090140 that incorporates this paper by reference into) .But probe can comprise the longer zone of the nucleotide sequence that is derived from the ParG gene, for example, and from about 50,60,75,100,200 or, 500 continuous nucleotides any in the ParG gene of mentioning.Preferably, probe should comprise the nucleotide sequence in one of high conservative zone of the catalyst structure domain of encoding, and described high conservative zone is by being identified comparing from the different PARG protein of animal.These zones also are present in the PARG protein from mouse ear mustard of evaluation, and comprise aminoacid sequence LXVDFANXXXGGG (corresponding to the 252nd amino acids to the 264 amino acids of the SEQ ID NO:1 of the WO 2004/090140 that incorporates this paper by reference into; X can be any amino acid), LXVDFANXXXGGGXXXXGXVQEEIRF (corresponding to the 252nd amino acids to the 277 amino acids of the SEQ ID NO:1 of the WO 2004/090140 that incorporates by reference this paper into) or LXVDFANXXXGGGXXXXGXVQEEIRFXXXPE (corresponding to the 252nd amino acids to the 282 amino acids of the SEQ ID NO:1 of the WO 2004/090140 that incorporates by reference this paper into), TGXWGCGXFXGD (corresponding to the 449th to 460 amino acids of SEQ ID NO:1) or TGXWGCGAFXGDXXLKXXXQ (corresponding to the 449th amino acids to the 468 amino acids of the SEQ ID NO:1 of the WO 2004/090140 that incorporates by reference this paper into). Other conservative region has aminoacid sequence DXXXRXXXXAIDA; (corresponding to the 335th amino acids to the 344 amino acids of the SEQ ID NO:1 of the WO 2004/090140 that incorporates this paper by reference into) or REXXKAXXGF; (corresponding to the 360th amino acids to the 369 amino acids of the SEQ ID NO:1 of the WO 2004/090140 that incorporates this paper by reference into) or GXXXXSXYTGY, (corresponding to the 303rd amino acids to the 313 amino acids of the SEQ ID NO:1 of the WO 2004/090140 that incorporates this paper by reference into) .Hybridization should preferably be carried out under rigorous condition.

" rigorous hybridization conditions " represented when using in this article: if at least 95% and preferred at least 97% sequence identity is arranged between probe and target sequence, will hybridize so usually.The example of rigorous hybridization conditions is: comprising 50% methane amide, 5 * SSC (150mM NaCl, 15mM trisodium citrate), 50mM sodium phosphate (pH 7.6), 5 * Denhardt ' s solution, 10% T 500 and 20 μ g/ml night incubation in the solution of sex change, excision vector DNA (for example salmon sperm DNA), then in about 65 ℃ the hybridization upholder washed for example about 10 minutes (twice) in 0.1 * SSC.Other hybridization and wash conditions are known, people such as Sambrook, and Molecular Cloning:A Laboratory Manual, second edition, Cold Spring Harbor, among the NY (1989), Chapter 11 example to some extent particularly.

Alternatively, also can separate ParG encoding gene or its part, wherein use the oligonucleotide comprise from least 20 continuous nucleotides of the nucleotide sequence of the PARG encoding gene of mentioning or its complementary sequence as primer by the technology of PCR-based.This type of primer can comprise the nucleotide sequence of the conservative region of encoding, and is as indicated above, or class nucleotide sequence complementation therewith.The oligonucleotide that can be used for this purpose can comprise the SEQ ID NO:5 of the WO 2004/090140 that incorporates this paper by reference into, by reference incorporate into the SEQ ID NO:6 of the WO 2004/090140 of this paper, by reference incorporate into this paper WO 2004/090140 SEQ ID NO:7 or incorporate the nucleotide sequence of SEQ ID NO:8 of the WO2004/090140 of this paper by reference into.Spendable oligonucleotide can also be a degeneracy, for example the Oligonucleolide primers of SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21 or SEQ ID NO:22, WO's 2004/090140 is whole, incorporates this paper by reference into.

Can for example following acquisition under strict annealing conditions from the specific PCR fragment of ParG gene: have the SEQ ID NO:5 of the WO 2004/090140 that incorporates this paper by reference into and incorporate the combination of oligonucleotide of nucleotide sequence of SEQ ID NO:6 of the WO 2004/090140 of this paper by reference into by use, for example use arabidopsis thaliana genomic dna or cDNA as template DNA, perhaps have the SEQ IDNO:7 of the WO 2004/090140 that incorporates this paper by reference into and incorporate the combination of oligonucleotide of nucleotide sequence of SEQ ID NO:8 of the WO 2004/090140 of this paper by reference into, for example use potato gene group DNA or cDNA as template DNA by use.

The functional PARG protein of separated sequence codified or its part.Preferably, separated sequence should comprise following nucleotide sequence, one or more in the high conservative zone of the proteinic catalyst structure domain of PARG that described sequence encoding is mentioned from other place.

But with regard to purpose of the present invention, separated sequence need not encoding function ParG protein, and also need not separate complete coding region.What in fact, need for the present invention only is: can based on from the endogenous ParG gene of plant through identifying or design or produce the mosaic gene that can produce the ParG inhibitory RNA through separation sequence.Can obtain some alternative approach, be used to produce this type of ParG inhibitory RNA molecules.

Clearly, when no matter when defining the nucleotide sequence of RNA molecule with reference to the nucleotide sequence of corresponding dna molecular, the thymus pyrimidine in the nucleotide sequence (T) all should be replaced by uridylic (U).And reference is that RNA or dna molecular will be clearly from the application's background.

When using in this article, specialize as nothing, " nucleotide sequence of PARP encoding gene or PARG encoding gene " is often referred to the nucleotide sequence of following DNA chain, described DNA chain on sequence corresponding to the nucleotide sequence of the mRNA molecule that comes from this type of PARP or PARG genetic transcription.

In a large amount of animals, identified the PARG encoding gene, described animal for example, Rattus norvegicus (accession number: NM_031339, NW_043030, AB019366), house mouse (accession number: NT_039598, NM_003631, AF079557), people (accession number: NT_017696, NM_003631, AF005043), ox (accession number: NM_174138, U78975), drosophila melanogaster (accession number: AF079556).

In plant, identified poly (ADP-ribose) glycogen lytic enzyme, this is to realize by the clone based on collection of illustrative plates to the wild type gene of inactivation in following mutant, in the transcribing of the clock control of the gene of described mutant in Arabidopis thaliana and be affected in relying on photoperiodic transformation (tej) from nourishing and growing to blooming.Can with accession number AF394690 from the Nucleotide database obtain gene nucleotide sequence (people such as Panda, 2002 Dev.Cell.3,51-61).

When using in this article, term " promotor " refers to during transcription initiation by any DNA of identification of dna dependent rna polysaccharase and combination (directly or indirectly).Promotor comprises transcription initiation site and at the binding site of transcription initiation factor and RNA polymerase, and can comprise some other sites (for example enhanser), can be in conjunction with gene expression regulation protein in these site.

(, regulation and control) given dna sequence dna (for example DNA of proteins encoded or the polypeptide) opportunity of transcribing and any DNA of level represented to relate to and driven and transcribe and control to term " regulation and control zone " when using in this article.For example, 5 ' regulation and control zone (or " promoter region ") is the dna sequence dna that is positioned at encoding sequence upstream (promptly 5 '), and it comprises promotor and 5 '-untranslated leader.3 ' regulation and control zone is the dna sequence dna that is positioned at encoding sequence downstream (that is, 3 '), and it comprises suitable Transcription Termination (and/or regulation and control) signal, and described signal comprises one or more poly-adenosine signals.

In one embodiment of the invention, promotor is a constitutive promoter.In another embodiment of the present invention, promoter activity strengthens (inducible promoters) by external or internal stimulation, and described stimulation is such as but not limited to hormone, chemical compound, mechanical power, abiotic or biological stress conditions.The activity of promotor can also be with time or space (tissue-specific promoter; Developmental regulation type promotor) mode is regulated and control.

With regard to purpose of the present invention, promotor be can be in plant expression promoter.When using in this article, term " can in plant expression promoter " expression can be controlled the dna sequence dna of transcribing in (initial) vegetable cell.This comprises the promotor of any plant origin, but also comprise the promotor that can instruct any non-plant source of transcribing in vegetable cell, promptly, some promotor of virus or bacterial origin, for example, CaMV35S (people such as Hapster, 1988), ground three mosaic virus promotor No 4 or No 7 (WO9606932) or T-DNA gene promoter, but also comprise tissue specificity or organ specific promoters, it includes but not limited to seed specific promoters (for example WO89/03887), organ-original hase specificity promoter (people such as An, 1996), stem specificity promoter (people such as Keller, 1988), leaf specificity promoter (people such as Hudspeth, 1989), mesophyll specificity promoter (for example photoinduction type Rubisco promotor), root-specific promoter (people such as Keller, 1989), stem tuber specificity promoter (people such as Keil, 1989), vascular tissue specificity promoter (people such as Peleman, 1989), (WO 89/10396 for stamen selective actuation, WO92/13956), cracking district's (dehiscence zone) specificity promoter (WO 97/13865) etc.

The method that is used for the mosaic gene introduced plant is well known in the art, and it comprises, and conversion, particle gun of Agrobacterium (Agrobacterium) mediation are sent, microinjection, the electroporation to intact cell, the protoplast transformation of polyoxyethylene glycol mediation, the electroporation to protoplastis, liposome-mediated conversion, the conversion that silicon-palpus mediates etc.What obtain by this way becomes sophisticated fertile plants through transformant is renewable then.

Also can comprise and to reduce the mosaic gene of WO 00/04173 described PARP genetic expression or will reduce the mosaic gene of WO 2004/090140 described PARG genetic expression according to vegetable cell of the present invention and department of botany.Can be for example by with transgenic plant of the present invention system and the transgenic plant hybridization that contains the mosaic gene that reduces PARP genetic expression, introduce above-mentioned other mosaic gene.Also can obtain transgenic plant cells or department of botany by mosaic gene of the present invention being introduced or being changed over to the transgenic plant cells (perhaps introduce conversely or change over to) that comprises the mosaic gene that reduces PARP genetic expression.Alternatively, can be encoded by a mosaic gene in PARP and PARG inhibitory RNA zone, and transcribe as a RNA molecule.

Before the coding as herein described-mosaic gene of miRNA molecule can experience as the mosaic gene of any introducing and transcribe back or Transcriptional Silencing.But this area can obtain to identify reticent method, comprises that the single stranded RNA molecule of determining 21-24nt is (preferably by identification Microrna and Microrna ^*The single stranded RNA of probe hybridization) existence.This type of reticent transgenic lines should preferably be avoided.

The all right instantaneous mode introduced plant cell of mosaic gene of the present invention (or its corresponding miRNA molecule) for example uses virus vector, for example the described viral rna vector of WO 00/63397 or WO02/13964.

A further object of the invention provides vegetable cell and the plant that contains with good grounds RNA molecule of the present invention or mosaic gene.Gamete, seed, embryo (zygotic embryo or somatic embryo), offspring or the crossbred of the plant that comprises mosaic gene of the present invention by traditional breeding way production also are included in the scope of the present invention.

Also can pass through traditional breeding technology, will be by plant and other plant hybridization of method acquisition as herein described, to obtain to comprise the stress tolerance progeny plants of mosaic gene of the present invention.

We believe, ways and means as herein described is applicable to all vegetable cells and plant, comprise dicotyledons and monocotyledons, and include but not limited to cotton, Btassica (Brasdica) vegetables, colea, wheat, corn or Zea mays, barley, clover, peanut, Sunflower Receptacle, rice, oat, sugarcane, soybean, turfgrass, barley, rye, Chinese sorghum, sugarcane, vegetables (comprise witloof, lettuce, tomato, zucchini, green pepper, eggplant, cucumber, melon, green onion, leek), tobacco, potato, preserved carrot, papaya, pineapple, mango, mouse ear mustard also is useful on gardening, plant (the willow of flower culture or forestry, Chinese fir, eucalyptus etc.).

This paper incorporates this paper into the degree of necessity by reference by all nucleotide sequences or the aminoacid sequence mentioned in the accession number of the obtainable database of the public.

When using in this article, " comprising " is appreciated that the existence of having pointed out feature, numeral, step or the component mentioned, but this does not get rid of the existence or the interpolation of one or more features, numeral, step or component or their group.Therefore, for example, comprise the nucleic acid of Nucleotide or aminoacid sequence or protein and can comprise more polynucleotide or amino acid except actual Nucleotide of mentioning or amino acid, that is, be embedded in the bigger nucleic acid or protein.The mosaic gene in the DNA zone that comprises on the function or be defined on the structure can comprise extra DNA zone etc.

Following non-limiting example has been described the ways and means that increases stress tolerance according to the present invention in plant.

Unless indicate in addition in an embodiment, all recombinant DNA technologies are all carried out according to standard scheme, people such as Sambrook (1989) Molecular Cloning:A Laboratory Manual is seen in the description of described standard scheme, second edition, Cold Spring Harbor Laboratory Press, NY and at people such as Ausubel (1994) Current Protocols in Molecular Biology, Current Protocols is in the volume 1 and volume 2 of USA.It is described to be used for the standard material of plant molecular work and the Plant Molecular Biology Labfax (1993) that method is seen R.D.D.Croy (BIOSScientific Publications Ltd (UK) and Blackwell Scientific Publications, UK combined publication).Other reference about standard molecular biological technique comprises: Sambrook and Russell (2001) Molecular Cloning:A Laboratory Manual, the third edition, ColdSpring Harbor Laboratory Press, NY, Brown (1998) Molecular BiologyLabFax, second edition, the volume 1 of Academic Press (UK) and volume 2.The standard material and visible Dieffenbach of method and Dveksler (1995) the PCR Primer:ALaboratory Manual that are used for the polymerase chain reaction, people such as Cold Spring Harbor Laboratory Press and McPherson. (2000) PCR-Basics:From Background to Bench, first version, Springer Verlag, Germany.

In specification sheets and embodiment, mention following sequence:

The cDNA of SEQ ID No:1:miRNA mb01-a01

The cDNA of SEQ ID No:2:miRNA mb01-b04

The cDNA of SEQ ID No:3:miRNA mb01-b09

The cDNA of SEQ ID No:4:miRNA mb01-h09

The cDNA of SEQ ID No:5:miRNA mb02-c04

The cDNA of SEQ ID No:6:miRNA mb02-g01

The cDNA of SEQ ID No:7:miRNA mb03-h06

The cDNA of SEQ ID No:8:miRNA mb04-d07

The cDNA of SEQ ID No:9:miRNA mb04-g09

The cDNA of SEQ ID No:10:miRNA mb06-c04

The cDNA of SEQ ID No:11:miRNA mb06-f06

The cDNA of SEQ ID No:12:miRNA mb06-h03

The cDNA of SEQ ID No:13:miRNA mb07-b12

The cDNA of SEQ ID No:14:miRNA mb07-c01

The cDNA of SEQ ID No:15:miRNA mb07-e03

The cDNA of SEQ ID No:16:miRNA mb08-e11

The cDNA of SEQ ID No:17:miRNA mb08-g02

The cDNA of SEQ ID No:18:miRNA mb08-h02

The cDNA of SEQ ID No:19:miRNA mb09-h05

The cDNA of SEQ ID No:20:miRNA mb10-b02

The cDNA of SEQ ID No:21:miRNA mb10-d05

The cDNA of SEQ ID No:22:miRNA mb10-g05

The cDNA of SEQ ID No:23:miRNA mb11-c05

The cDNA of SEQ ID No:24:miRNA mb11-d04

The cDNA of SEQ ID No:25:miRNA mb11-e04

The cDNA of SEQ ID No:26:miRNA mb13-c11

The cDNA of SEQ ID No:27:miRNA mb14-a09

The cDNA of SEQ ID No:28:miRNA mb15-a06

The cDNA of SEQ ID No:29:miRNA mb16-g11

The cDNA of SEQ ID No:30:miRNA mb17-e10

The cDNA of SEQ ID No:31:miRNA mb18-b02

The cDNA of SEQ ID No:32:miRNA mb18-e02

The cDNA of SEQ ID No:33:miRNA mb18-h12

The cDNA of SEQ ID No:34:miRNA mb19-d08

The cDNA of SEQ ID No:35:miRNA mb19-g05

The cDNA of SEQ ID No:36:miRNA mb20-c07

SEQ ID No:37: the nucleotide sequence of the mosaic gene of coding miRPARP and the variant in miRPARPm2 and miRPARPm3 thereof

The nucleotide sequence of SEQ ID No:38:miRPARP7

SEQ ID No:39: the nucleotide sequence of preceding-Microrna 398a

SEQ ID No:40: transformed nucleotide sequence with the preceding-Microrna 398a that is processed into miRPARP7

The nucleotide sequence of SEQ ID No:41:miRPARP7

SEQ ID No:42: transformed nucleotide sequence with the preceding-Microrna 398a that is processed into miRPARP2-8

Embodiment

Embodiment 1: to relating to the evaluation of the novel miRNA molecule of inanimate stress response in the plant

According to this area standard method, from unscared mouse ear mustard seedling in 2 age in week or from experiencing two cDNA libraries of 2 mouse ear mustard seedling structures in age in week of high optical condition.Only use and comprise the RNA molecule fraction that length is about 15 to 30 Nucleotide.

The clone checks order to random cdna.At first filter these sequences, contain corresponding to those of the cDNA of the long RNA of about 20 to 25 Nucleotide only to keep.Residue sequence and mouse ear mustard genome sequence (using BLAST) are compared, to discard the sequence that has sequence identity with ribosome-RNA(rRNA) sequence, tRNA sequence, chloroplast(id) or mitochondrial RNA(mt RNA) sequence.This analysis has also disclosed at residue sequence, the corresponding locus in the arabidopsis gene group that potential miRNA molecule can derive from.

Use the FastA algorithm at Arabidopis thaliana coding region database, can identify the have sequence similarity possible target sequence of (but allowing mispairing).

In next procedure, use mFold, analyze genome area with regard to secondary structure corresponding to the genome position of the small RNA molecular that keeps, with before identifying-the miRNA spline structure.

At remaining potential miRNA structure, analyze coerce with non-stress conditions under the expression pattern of respective target sequence, have than non-those miRNA structures of coercing the target gene of plant differential expression in coercing plant and be retained down.These miRNA and corresponding target gene are shown in Table 1.

Make up the mosaic gene of the preceding-miRNA structure of encoding, described structure comprises the miRNA sequence of table 1, and described mosaic gene is expressed under the control of strong constitutive promoter.Structure comprises the transgenic plant of these mosaic genes, analyzes the influence to the transgenic plant stress tolerance.

Table 1

Embodiment 2: the mosaic gene that makes up the synthetic miRNA molecule of the endogenous PARP gene of coding target

Based on preceding-miRN171 support, design synthetic before-the miRNA nucleotide sequence, its comprise with the parp1 of mouse ear mustard and parp2 coding region between the 21 nucleotide sequence complementary miRNA sequences of conservative (identical).This nucleotide sequence is positioned at so-called PARP label construction territory.For reaching this purpose, use and PARP1/PARP2 conserved sequence complementary nucleotide sequence (Fig. 1; Figure B) replace before-miRNA nucleotide sequence (Fig. 1 of miR171 support; Scheme A), and containing miRNA ^*Making corresponding Nucleotide in the chain changes.At last, at miRNA ^*Introduce two Nucleotide in the chain and change, cause the mispairing in the dsRNA stem, natural before-two projection (Fig. 1 of position introducing of projection take place among the miRNA171; Figure C).Use standard technique to synthesize the corresponding D NA nucleotide sequence that produces.

Use standard recombinant technology makes up the mosaic gene with following dna fragmentation that effectively links to each other:

CaMV35S promotor (SEQ ID NO:37 is from nt1 to 531)

Untranslated leader (Cab22L; SEQ ID NO:37 is from nt531 to 591)

Comprise modified preceding-mi171 DNA zone of (comprising miRPARP (SEQ ID NO:37 is from nt592 to 737))

3 ' stub area of CaMV 35S transcript (SEQ ID NO:37 is from nt737 to 958).

Make up similar mosaic gene, wherein the nucleotide sequence of miRPARP is through modifying, to introduce mispairing (seeing Fig. 2, figure B and C) between miRNA molecule and target PARP1/PARP2 nucleotide sequence.Following Nucleotide is substituted:

In miRPARP2m:

nt?619：G->A

nt?630：G->A

nt?695：C->T

nt?706：C->T

In miRPARP3m:

nt?619：G->A

nt?620：T->G

nt?630：G->A

nt?695：C->T

nt?705：A->C

nt?706：C->T

Mosaic gene is introduced mouse ear mustard plant, produce the transgenic lines that comprises different mosaic genes.Order was grown for two weeks under optimal conditions from different transgenic lines and from the seedling of the control plant of unconverted and positive control stress-tolerance system, then they was imposed 3 days high light and coerced.Every system measures the weight of 25 strain seedling, and this is shown in Table 2.

The high light of table 2:miRPARP is coerced experiment:

Department of botany

Weight

Col-0 (contrast)	400
Col-0 (contrast)	400	MiRPARPm3 is 1	460
MiRPARPm2 is 1	593	MiRPARPm3 is 1	460
MiRPARPm2 is 1	593	MiRPARP is 1	595
MiRPARPm3 is 1	627	MiRPARP is 1	595
MiRPARPm3 is 1	627	Positive control 1 (WO 00/04173 described transgenic lines)	641
MiRPARP is 2	757	Positive control 1 (WO 00/04173 described transgenic lines)	641
MiRPARP is 2	757	MiRPARP is 3	759
Positive control 2 (WO 00/04173 described transgenic lines)	769	MiRPARP is 3	759
Positive control 2 (WO 00/04173 described transgenic lines)	769	MiRPARP is 4	798

In growth under the high light stress conditions after 3 days, all transgenic lines that comprise miRNA all show than the remarkable higher weight of the control series of unconverted.The increase of stress tolerance is reduced similar that the PARP gene is obtained to the dsRNA that uses the described coding mosaic gene of WO00/04173.

Use CTAB to separate total nucleic acid in a conventional manner, on 15% acrylamide/urea gel, separate, and with discerning Microrna chain or complementary Microrna ^*The LNA oligonucleotide probe of chain is surveyed.Can not detect the oligonucleotide of 21nt, but in different transgenic lines, identified 80nt long through the processing RNA sequence, itself and the probe hybridization of identification miRPARP2.These results most possibly show the problem that preceding-Microrna is correctly processed.

Based on preceding-miRNA398a support, design another synthetic before-the miRNA nucleotide sequence, its comprise with the parp1 of mouse ear mustard and parp2 coding region between the 21 nucleotide sequence complementary miRNA sequences of conservative (identical).For reaching this purpose, use the miRNA nucleotide sequence (Fig. 4, left figure) of replacing preceding-miR398a support with PARP1/PARP2 conserved sequence complementary nucleotide sequence (Fig. 3), and containing miRNA ^*Making corresponding Nucleotide in the chain changes.Use standard technique to synthesize the corresponding D NA nucleotide sequence (SEQ ID NO:40) that produces.

CaMV35S promotor (SEQ ID NO:37 is from nt1 to 531)

Untranslated leader (Cab22L; SEQ ID NO:37 is from nt 531 to 591)

Comprise modified preceding-mi398a DNA zone of (comprising miRPARP7 (SEQ ID NO:40))

3 ' stub area of CaMV35S transcript (SEQ ID NO:37 is from nt 737 to 958).

Mosaic gene is introduced mouse ear mustard plant, produce the transgenic lines that comprises different mosaic genes.

Use CTAB to separate in a conventional manner, on 15% acrylamide/urea gel, separate from different transgenic lines total nucleic acid, and with discerning Microrna chain or complementary Microrna ^*The LNA oligonucleotide probe of chain is surveyed.The long strand Nucleotide of 21 and 24 Nucleotide have been identified, itself and arbitrary Microrna probe and Microrna ^*Probe hybridization.This shows that preceding-miRPARP7 is not processed as natural miRNA.In addition, this may show, transcribing and/or post-transcriptional silencing the mosaic gene of the coding miRNA that introduces taken place.

Based on preceding-miRNA398a support, design another synthetic before-the miRNA nucleotide sequence, its comprise with to specific 21 the nucleotide sequence complementary miRNA sequences of the parp2 coding region of mouse ear mustard.For reaching this purpose, use the miRNA nucleotide sequence (Fig. 6, left figure) of replacing preceding-miRNA398a support with PARP2 specific sequence complementary nucleotide sequence (Fig. 5), containing miRNA ^*Making corresponding Nucleotide in the chain changes.Use standard technique to synthesize the corresponding D NA nucleotide sequence (SEQ ID NO:42) that produces.

CaMV35S promotor (SEQ ID NO:37 is from nt 1 to 531)

Untranslated leader (Cab22L; SEQ ID NO:37 is from nt 531 to 591)

Comprise modified preceding-mi398a DNA zone of (comprising miRPARP2-8 (SEQ ID NO:42))

3 ' stub area of CaMV 35S transcript (SEQ ID NO:37 is from nt 737 to 958).

Use CTAB separates the total nucleic acid from different transgenic lines in a conventional manner, on 15% acrylamide/urea gel, separates, and with discerning Microrna chain or complementary Microrna ^*The LNA oligonucleotide probe of chain is surveyed.The results are shown among Fig. 7.When hybridizing (figure A) with the probe of identification Microrna, but not with discerning Microrna ^*Probe when hybridizing, identified 21 strand Nucleotide that Nucleotide is long.This shows that preceding-miRPARP2-8 is correctly to be processed with the similar mode of natural miRNA.

Use the primer of specific amplification AtPARP2 nucleotide sequence, carry out RT-PCR at mRNA from control series (pTMBO009) or transgenic lines (mosaic gene that comprises the miRPARP2-8 that encodes) (pTMBO024-1 and pTMBO024-3) acquisition.Obtain mRNA from the plant of being coerced that (HL) grows growing plants under the normal light condition or under high optical condition.Fig. 8 is the diagram to the level of the parp2 mRNA that does not detect in the homology under different condition.PTMBO024 show normal and be subjected to the level of the reduction of parp2 mRNA under the stress conditions, pTMO024-3 only then to show under normal operation the level of parp2 mRNA reduces.

Make the plant of pTMO024-1 system experience multiple inanimate stress conditions.Be subjected to not existing of PARP2 induced expression under the stress conditions, or the expression of PARP2 reduces, shown high stress resistance.

Fig. 9 shows pri-miR171 (Fig. 9 A), come from the secondary structure of primary transcribe of pri-miRPARP7 (Fig. 9 B) of pri-miR171 and each amplifier section (Fig. 9 C and D) around preceding-miR171 and the preceding-miRPARP7 processing site.Figure 10 shows pri-miR398 (Figure 10 A), come from the secondary structure of primary transcribe of pri-miRPARP2-8 (Figure 10 B) of pri-miR398 and each amplifier section (Figure 10 C and D) around preceding-miR398 and the preceding-miRPARP2-8 processing site.In both cases, the secondary structure of the primary transcribe of wild-type support and modified support is all quite different; But (1,2 among two width of cloth figure) are then relatively very conservative for the secondary structure of preceding-miRNA substructure.But, the announcement that overhauls to secondary structure (Fig. 9 C and D and 10C and D) around preceding-miRNA processing site: before transforming-the miR171 support to be when mixing the miRPARP7 sequence, single stranded RNA structure (12) and (13) and single stranded RNA structure (4) and (9) and double-stranded RNA stem (3,8) with respect to existing change of banded distance between preceding-miRNA processing site (10,11).Especially, double-stranded RNA stem (8) length among the pri-miPARP7 increases, and this is because the existence of part complementary nucleotide sequence (6) and (7).This may cause the processing to preceding-miRNA in the position different with original pri-mRN171 in the synthetic molecule, this so that may have influence on before synthetic-miRNA is to the processing of the miRNA of correct design.On the contrary, in pri-miR398 and deutero-pri-miRPARP2-8, the relative position that is attached between single-stranded structure (5,13,6 and 8) and double-stranded RNA stem (4), (7) is very conservative.Before pri-miRPARP2-8 seems correctly to be processed as-and miRPARP2-8 and miRPARP2-8, this declares publicly and keeps banded importance between single stranded RNA structure and the double-stranded RNA stem in the preceding-miRNA process zone that structurally is similar to used pri-miRNA support.

Sequence table

＜110〉Bayer Biochemical Sciences S.A. (Bayer BioScience N.V.)

Vib Vzw (Vlaams Interuniversitair Instituut voorBiotechnologie)

State-run Ghent, Belgium university (Universiteit Gent)

M win thatch (Bots, Marc)

M plum thatch widen (Metzlaff, Michael)

＜120〉Xin Ying stress-related microRNA molecules and uses thereof

<130>BCS?06-2006

<150>EP?06009836.5

<151>2006-05-12

<150>US60/808036

<151>2006-05-24

<160>42

＜170〉PatentIn version 3 .3

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＜223〉mosaic gene of preceding-miRNA of coding target PARP

<220>

<221>misc_feature

<222>(1)..(531)

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<220>

<221>misc_feature

<222>(532)..(591)

＜223〉untranslated leader sequence (Cab221)

<220>

<221>misc_feature

<222>(592)..(736)

＜223〉preceding miRNA

<220>

＜221〉variation

<222>(619)..(619)

＜223〉G to A among the miPARP2m

<220>

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<222>(619)..(619)

＜223〉G to A among the miRPARP3m

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<222>(620)..(620)

＜223〉T to G among the miRPARP3m

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<222>(630)..(630)

＜223〉G to A among the miPARP2m

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<222>(630)..(630)

＜223〉G to A among the MiRPARP3m

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<222>(689)..(709)

<223>miPARP

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<222>(696)..(696)

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<222>(696)..(696)

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<222>(706)..(706)

＜223〉A to C among the miRPARP3m

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<222>(707)..(707)

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<222>(737)..(958)

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<222>(34)..(54)

<223>miR398a*

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<222>(98)..(117)

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<222>(34)..(54)

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<222>(98)..(118)

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＜223〉Microrna miRPARP2-8

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＜223〉pre-Microrna miRPARP2-8

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<400>42

Claims

1. separated RNA molecule, it can instruct the cutting to target sequence, and described RNA molecule comprises nucleotide sequence arbitrary among SEQ ID NO:1 to the SEQ ID NO:36 or its can instruct variant to the cutting of described target sequence.

2. separated dna molecular, it can instruct the cutting to target sequence, and described dna molecular comprises nucleotide sequence arbitrary among SEQ ID NO:1 to the SEQ ID NO:36 or its can instruct variant to the cutting of described target sequence.

3. mosaic gene, it comprises the following DNA zone that effectively links to each other:

A. can be in plant expression promoter;

The b.DNA zone, described DNA zone is processed into miRNA at the introduced plant cell and after transcribing therein, described miRNA can instruct the cutting to target sequence, and described DNA zone comprises nucleotide sequence arbitrary among SEQ ID NO:1 to the SEQ ID NO:36 or its can instruct variant to the cutting of described target sequence; And

C. randomly, 3 ' the DNA zone that relates to Transcription Termination and poly-adenosineization.

4. the mosaic gene of claim 4, wherein, described can expression promoter be constitutive promoter or tissue-specific promoter or inducible promoters in plant.

5. the mosaic gene of claim 4, wherein, described can expression promoter be the promotor of rna plymerase ii identification in plant.

6. the mosaic gene of claim 4, wherein, described can expression promoter be the promotor of rna plymerase iii or rna plymerase i or rna plymerase i V identification in plant.

7. the vegetable cell that comprises mosaic gene any among the claim 4-6.

8. the plant that constitutes by the vegetable cell of claim 7 basically.

9. method, it is used for reducing plant PARP or PARG expression levels, or is used to increase the resistance of plant for unfavorable growth conditions, and described method comprises the steps:

A. introduce mosaic gene in the cell of described plant, described mosaic gene comprises the following DNA zone that effectively links to each other:

I. can be in plant expression promoter;

The ii.DNA zone, described DNA zone is processed into miRNA at the introduced plant cell and after transcribing therein, and described miRNA can discern and instruct the cutting to the mRNA of endogenous PARP gene of described plant or ParG gene; And

Iii. randomly, 3 ' the DNA zone that relates to Transcription Termination and poly-adenosineization.

10. according to the method for claim 9, wherein, described DNA zone comprises the basic complementary nucleotide sequence of nucleotide sequence with at least 21 continuous nucleotides of the described endogenous PARP gene of described plant or ParG gene, described 21 continuous nucleotides produce described miRNA, and condition is to allow one or more following mispairing:

A. the mispairing in Nucleotide that described miRNA5 ' is terminal and the described RNA molecule between the corresponding nucleotide sequence;

B. the mispairing between the corresponding nucleotide sequence in the arbitrary and described RNA molecule in the Nucleotide in the 1st of described miRNA the to the 9th;

C. three mispairing between the corresponding nucleotide sequence in the arbitrary and described RNA molecule in the Nucleotide in the 12nd of described miRNA the to the 21st, prerequisite is to be no more than two continuous mispairing.

11. according to the method for claim 9 or 10, wherein, described miRNA comprises the nucleotide sequence of SEQ ID NO:37 from 709 Nucleotide of the 689th Nucleotide to the, or the nucleotide sequence of the nucleotide sequence of SEQ ID NO:38 or SEQ ID NO:41.

12. according to method any in the claim 9 to 11, wherein, described mosaic gene coding comprises the primary transcribe of preceding-miRNA, when in vegetable cell when processed described before-miRNA produces described miRNA, described before-miRNA be derived from naturally occurring before-miRNA, described primary transcription instinct adopts secondary RNA structure, and it comprises single stranded RNA structure and double-stranded RNA stem and preceding _-mThe iRNA processing signal, wherein, described before-described single stranded RNA structure in the miRNA processing signal zone and the binding between the described double-stranded RNA stem be similar to described naturally occurring before _-mBinding described in the iRNA between single stranded RNA structure described in preceding-miRNA processing signal zone and the described double-stranded RNA stem.

13. according to the method for claim 12, wherein said before-miRNA be derived from before-miR398.

14. according to method any in the claim 9 to 12, wherein, described mosaic gene also comprises the nucleotide sequence of 147 Nucleotide of the 119th Nucleotide to the of the nucleotide sequence of 97 Nucleotide of the 55th Nucleotide to the of the nucleotide sequence of 33 Nucleotide of the 1st Nucleotide to the of SEQ ID NO:41 and SEQ ID NO:41 and SEQID NO:41.

15. according to method any in the claim 9 to 14, wherein, described mosaic gene comprises the nucleotide sequence of SEQ ID NO:42.

16. according to method any in the claim 9 to 15, wherein, described can expression promoter be constitutive promoter or tissue-specific promoter or inducible promoters in plant.

17. according to method any in the claim 9 to 15, wherein, described can expression promoter be the promotor of rna plymerase ii identification in plant.

18. according to method any in the claim 9 to 15, wherein, described can expression promoter be the promotor of rna plymerase iii or rna plymerase i or rna plymerase i V identification in plant.

19. according to method any in the claim 9 to 18, show the tolerance that low or high temperature, arid, highlight strength, chemical pollution, water logging, high salinity, highlight strength, high UV radiating are increased when wherein, described plant is compared with the isogenic line that does not comprise described mosaic gene.

20. mosaic gene, it is as described in the claim 9 to 18 any one.

21. vegetable cell, it comprises the mosaic gene according to claim 20.

22. plant, it is made of the vegetable cell of claim 22 basically.

23. Microrna, it is as described in the claim 9 to 18 any one.

24. according to the Microrna of claim 23 or be used to increase the purposes of the stress tolerance of vegetable cell or plant according to the mosaic gene of claim 20.

25. method, it is used for reducing miRNA for the influence that reduces at the vegetable cell expression of target gene, and described method comprises the steps:

A. provide the excessive RNA substrate of being discerned and cutting by miRNA to described vegetable cell, described RNA substrate is discerned and is cut by described miRNA.

26. the method for claim 25, wherein, described excessive RNA substrate provides by expressing following mosaic gene, and described mosaic gene comprises the following dna fragmentation that effectively links to each other:

A. can be in plant expression promoter;

B.DNA zone, described DNA zone produce the RNA molecule after introducing and transcribing, described RNA molecule comprises the nucleotide sequence of the miRNA cutting that is comprised in the RISC protein complexes;

27. the method for claim 26, wherein, described RNA molecule comprises and the basic complementary nucleotide sequence of described miRNA molecule, and condition is to allow one or more following mispairing:

28. any one method in the claim 25 to 27, wherein, the influence of the described minimizing of described miRNA causes expression arbitrary in the target gene of described miRNA to increase.