CN101225433A - Plant miRNA chip and uses thereof - Google Patents
Plant miRNA chip and uses thereof Download PDFInfo
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- CN101225433A CN101225433A CNA2007100365763A CN200710036576A CN101225433A CN 101225433 A CN101225433 A CN 101225433A CN A2007100365763 A CNA2007100365763 A CN A2007100365763A CN 200710036576 A CN200710036576 A CN 200710036576A CN 101225433 A CN101225433 A CN 101225433A
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Abstract
The invention discloses a miRNA chip, comprising a solid phase support and a plurality of oligonucleotide probes orderly fixed on the solid phase support, which is characterized in that: the sequence showed by the SEQ ID NO: 1-SEQ ID NO: 137 is specifically in connection with the oligonucleotide probe; and the reagent kit containing the chip and the method utilizing the miRNA chip to detect the miRNA expression profile in the plant are disclosed. The nucleic acid trigeminy probes and nucleic acid quadruple probes has the advantages that: a class of new miRNA separated from the plant is prepared to the chip, so as to provide convenient way for researching the miRNA expression profile and the regulation for the plant from the miRNA.
Description
Technical field
The invention belongs to biotechnology and phytology field, more specifically, the present invention relates to a kind of Mirnas of plant chip and uses thereof.
Background technology
MiRNA (micro RNA) is one group of not short sequence single stranded RNA of coded protein, is about 22nt.MiRNA gene mainly single or cluster ground is positioned at genomic non-coding region.The miRNA that present people have found is about thousands of, most of Unknown Function.According to present studies show that, miRNA carries out a kind of back regulation mechanism of transcribing, and plays an important role in the protein expression process.MiRNA is by the mode and corresponding regional combination of mRNA of incomplete base complementrity, thus the translation of arrestin matter; In certain plants, its mRNA that can also degrade.A common gene can be regulated by a plurality of miRNA, and a miRNA also can regulate a plurality of mRNA simultaneously.
MiRNA expresses conservative property, sequential and the tissue specificity with height.Studies show that miRNA has participated in nearly all vital movement, and play an important role, in animal, comprising: the generation of cell development, hematopoiesis, metabolism of fat, organ generation, apoptosis, cell proliferation and differentiation and tumour.In addition, in some virus, also find have miRNA to express.
In vegetable cell, the miRNA precursor of transcribing formation at first is folded into loop-stem structure, and the effect down cut at DCL1 forms sophisticated miRNA then.At different plant tissues, organ and different developmental stages, the express spectra of miRNA is different, will play huge pushing effect to the research of miRNA biological function to the research of miRNA express spectra.
Although in organism, found some miRNA at present, yet these miRNA only account for a part quite few among the miRNA that exists in the organism, and mostly are Unknown Function for these miRNA.And the existing method that adopts Northern hybridization or miRNA to clone to the research of miRNA express spectra needs a large amount of total RNA samples, and wastes time and energy more, and progress is slow.
Therefore, this area presses for separates miRNA further, and what is more important exploitation suitable way, with the express spectra of research miRNA and they regulating effect for organism.
Summary of the invention
The object of the present invention is to provide a kind of miRNA chip, this chip can be used for detecting the express spectra of miRNA or the miRNA regulative mode for growth and development of plants.
In a first aspect of the present invention, a kind of miRNA chip is provided, described miRNA chip comprises:
Solid phase carrier; And
Be fixed on the oligonucleotide probe on the described solid phase carrier in order, described oligonucleotide probe is specifically corresponding to the part or all of sequence shown in the SEQ ID NO:1-SEQ ID NO:137.
In another preference of the present invention, described oligonucleotide probe contains:
Complementary land; And/or
The joining region that links to each other with solid phase carrier.
In another preference of the present invention, described joining region is optional, also is that described complementary land can directly link to each other with solid phase carrier.
In another preference of the present invention, described " specifically corresponding to " refers under tight hybridization conditions, described probe can with the hybridization of the nucleotide sequence of SEQ ID NO:1-SEQ ID NO:137; More preferably, the nucleotide sequence of the sequence of the complementary land of described probe and SEQ ID NO:1-SEQ ID NO:137 is complementary fully.
In another preference of the present invention, the complementary land of described oligonucleotide probe is: with the sequence that is selected from the sequence complementation shown in the SEQ ID NO:1-SEQ ID NO:137 (preferred, as to be complete complementation).
In another preference of the present invention, described oligonucleotide probe is by 20-80 based composition.
In another preference of the present invention, be used to detect the express spectra of miRNA; Or be used to detect the regulative mode of miRNA for plant-growth or growth.
In a second aspect of the present invention, a kind of method that detects miRNA express spectra in the plant is provided, comprise step:
(1) provides separation from the RNA of plant sample, on described RNA, marker is set;
(2) RNA with (1) contacts with described chip, makes the oligonucleotide probe generation hybridization on described RNA and the solid phase carrier, thereby forms " oligonucleotide probe-RNA " binary complex on solid phase carrier; With
(3) detect the marker of the binary complex of (2) formation, thereby determine the express spectra of corresponding miRNA in the plant.
In another preference of the present invention, in step (1), before being set, marker also comprises step: enrichment small fragment RNA from the RNA sample.
In another preference of the present invention, the length of described small fragment RNA is 10-100bp (being more preferably 15-60bp).
In another preference of the present invention, described marker is selected from: fluorescein or derivatives thereof (as FITC, Cy3, Cy5 etc.), digoxin (DIG), vitamin H (Bio), alkaline phosphatase (AP), horseradish peroxidase (HRP).
In another preference of the present invention, described marker is connected with the 3 ' end of described RNA.
The third aspect in invention provides a kind of test kit, contains described miRNA chip in the described test kit.
Others of the present invention are because the disclosure of this paper is conspicuous to those skilled in the art.
Description of drawings
Fig. 1 has shown miRNA expression of gene pattern in the seed of spending 11 kinds in the paddy rice, leaf, root, endosperm, the embryo; Among the figure, color board is being indicated the expression intensity of miRNA, and the variation that grows from weak to strong that shown-2.0 (blueness)-0 (black)-2.0 (yellow) expression miRNA expresses is expressed strong more the closer to 2.0 expressions.Express weak more the closer to-2.0 expressions.
Fig. 2 has shown a kind of crossbreeding effect figure of chip of the present invention.
Embodiment
The inventor is through extensive and deep research, from the seed of the different developmental phases of paddy rice, separate first and obtain the new miRNA of a class, developed a kind of miRNA chip at these miRNA, this miRNA chip can be used for studying the express spectra of miRNA and the miRNA regulating effect for variety of event in the growth and development of plants, and it is few, easy to operate efficient to consume sample.Finished the present invention based on this.
As used herein, described " plant (or crop) " includes but not limited to: grass (as paddy rice), leguminous plants etc.In addition, described plant also can comprise forestry crop, arbor, flower plant etc.Preferably, described plant is meant anyly carries miRNA provided by the present invention (its sequence is shown in SEQ ID NO:1-SEQ ID NO:137) or carries the miRNA that has higher homology (as having 80% homology or higher) with described miRNA, or the expression of its mRNA can be by the general name of the plant that miRNA provided by the present invention influenced.
As used herein, described " miRNA " is meant a kind of RNA molecule, processed from the transcript that can form the miRNA precursor.Sophisticated miRNA has 18-26 Nucleotide (general about 22 Nucleotide) usually, yet does not also get rid of the miRNA molecule with other number Nucleotide.MiRNA can be detected by the Northern trace usually.The miRNA of plant origin is the miRNA that is obtained by precursor miRNA processing in vegetable cell.
As used herein, " Mirnas of plant express spectra " is meant the displaying collection of illustrative plates that shows the expression of miRNA in plant tissue or the cell.
As used herein, the little RNA of plant (small RNA) is meant the non-coding RNA of length between 18-26 in the vegetable cell, and they play a role with the form of RNA; MiRNA is a kind of of little RNA, can identify a part of miRNA from described little RNA, and the feature of miRNA is to result from a transcript, and this transcript is folded to form loop-stem structure, and miRNA is from a certain side in the stem structure.
As used herein, " small fragment RNA " is meant the length that the is separated to mixture at the RNA of 10-100bp molecule from the total RNA of plant.
miRNA
The invention provides the miRNA that a class is found from plant, Mirnas of plant can be from precursor miRNA processing, and described precursor miRNA can be folded into a kind of stable stem ring (hair clip) structure, and described loop-stem structure length is generally between 60-330bp.Usually, precursor miRNA can be sheared into sophisticated miRNA molecule in vegetable cell.
The inventor adopts extensive parallel signal sequencing system, separates, identifies a large amount of new miRNA from the seed of paddy rice different developmental phases.
Particularly, the inventor spends from the wild-type paddy rice and extracts the little RNA (small RNA) that total RNA isolates 18-26nt in 11 the seed, after little RNA is cloned into carrier, obtain the Signature sequence with the MPSS technical Analysis, masking may be the sequence in joint source, and the sequence that credibility is lower removal.
Then, the inventor navigates to the Signature sequence on the rice chromosome with relatively more all Signature sequence and the rice genome sequences (TIGR) of blast program (NCBI), rejects the sequence that does not have coupling on genome.With Rfam database (Sanger) is reference, removes rRNA, tRNA, snRNA and snoRNA etc.In order to differentiate the miRNA in the remaining sequence, on genome, choose the precursor sequence that comprises these sequences with the window (window) of 400bp, with its secondary structure of RNAfold software prediction.Satisfy simultaneously following standard for miRNA: 1. miRNA is positioned on a certain arm of front body structure (5 ' or 3 '), and the front body structure free energy is minimum; 2. be that the mispairing number must not be greater than 7 among the 25nt at center with miRNA; 3. there is not big ring texture on the stem of front body structure.
The sequence of the miRNA that these are identified out is shown in SEQ ID NO:1-SEQ ID NO:137; The sequence of described miRNA corresponding precursor miRNA is shown in SEQ ID NO:138-SEQ ID NO:274.
The miRNA chip
Because it is very low to detect the expression efficient of single miRNA in plant, therefore, the inventor designs suitable probe according to described miRNA, is fixed on the solid phase carrier, forms " oligonucleotide arrays ".Described " oligonucleotide arrays " is meant have the addressable point array of (promptly with distinctive, addressable address is the position of feature), and a coupled characteristic oligonucleotide is all contained in each addressable point.As required, oligonucleotide arrays can be divided into a plurality of inferior battle arrays.
MiRNA chip of the present invention comprises solid phase carrier and is fixed on oligonucleotide probe on the described solid phase carrier in order that described oligonucleotide probe is specifically corresponding to the sequence shown in the SEQ ID NO:1-SEQ ID NO:137.
As optimal way of the present invention, described probe comprises complementary land, the nucleic acid array complementation (preferred, as to be complete complementation) of the sequence of described complementary land and SEQ ID NO:1-SEQ ID NO:137.
As optimal way of the present invention, also contain the joining region on the described probe, described joining region is used to make probe stably to be fixed on solid phase carrier, and described joining region can be positioned at the two ends of complementary land, perhaps also can only be positioned at an end of complementary determining region.Certainly, under situation about allowing, also can select the mode that complementary determining region directly is connected with solid phase carrier.
As more preferably mode of the present invention, described joining region also has the function of regulating the GC ratio, is used to regulate the GC content of probe, thereby makes that the GC ratio of probe is 20-80%, and preferred GC ratio is 40-60%.
Therefore, probe of the present invention is by 20-80 (being more particularly 25-60) based composition continuously.In order to strengthen the intensity of detection signal, improve the accuracy rate of detected result, the above complementary land of probe preferably is positioned at the middle part of place probe.Described probe can also comprise glycosyl modified at its 5 ' end.
Described solid phase carrier can adopt the various common used materials in gene chip field, such as but not limited to nylon membrane, and the slide of slide of modifying through active group (as aldehyde radical, amino, the fine acidic group of different sulphur etc.) or silicon chip, unmodified, plastic sheet etc.
The preparation of miRNA chip of the present invention also can be adopted the conventional manufacture method of other biochip.For example, if what solid phase carrier adopted is to modify slide or silicon chip, 5 ' end of probe contains amido modified poly-dT string, oligonucleotide probe can be mixed with solution, adopt point sample instrument that its point is being modified on slide or the silicon chip then, be arranged in predetermined sequence or array, spending the night by placement then fixes, and just can obtain miRNA chip of the present invention.If it is amido modified that nucleic acid does not contain, then its preparation method also can reference: Wang Shenwu chief editor's " gene diagnosis technology-on-radiation operational manual "; J.L.erisi, V.R.Iyer, P.O.BROWN.Exploring the metabolic and genetic control of gene expression on a genomic scale.Science, 1997; 278:680 and Ma Li people, the Jiang Zhonghua chief editor. biochip. Beijing: Chemical Industry Press, 2000,1-130.
As an aspect, the invention provides a kind of method by miRNA express spectra in the miRNA chip detection plant, comprise step:
(1) provides separation from the RNA of plant sample, on described RNA, marker is set;
(2) RNA with (1) contacts with described chip, makes the oligonucleotide probe generation hybridization on described RNA and the solid phase carrier, thereby forms " oligonucleotide probe-RNA " binary complex on solid phase carrier;
(3) detect the marker of the binary complex of (2) formation, thereby determine the express spectra of corresponding miRNA in the plant.
The method of extracting RNA from plant tissue is a method well known to those skilled in the art.Such as adopting the Trizol method.
Preferred, in step (1), after from plant tissue, isolating the RNA sample, the RNA sample is suitably handled, have the RNA of certain-length with enrichment, described length is between 10-100 (small fragment RNA) generally.Through after the above-mentioned processing, utilize these small fragment RNAs to carry out follow-up hybridization, can improve the accuracy that chip is caught miRNA like this.Those skilled in the art can isolate the RNA with certain fragment length easily, such as adopting gel electrophoresis to separate.
It also is method well known to those skilled in the art that RNA is carried out mark, and it can be by the method realization of adding when hybridizing with RNA specificity bonded marker, and described marker is such as being labelling groups.Described labelling groups includes but not limited to: digoxin molecule (DIG), biotin molecule (Bio), fluorescein and the biomolecules of deriving (FITC etc.) thereof, other fluorescence molecule (as Cy3, Cy5 etc.), alkaline phosphatase (AP), horseradish peroxidase (HRP) etc.These marks and marking method thereof all have been routine techniques well-known in the art, also can be with reference to Wang Shenwu chief editor's " gene diagnosis technology-on-radiation operational manual "; J. Sa nurse Brooker, D.W. Russell chief editor, " molecular cloning experiment guide ", Science Press, 2002; Horse stands the people, the Jiang Zhonghua chief editor. biochip. and Beijing: Chemical Industry Press, 2000,1-130.
When above-mentioned RNA and miRNA chip are hybridized, can earlier miRNA chip and prehybridization damping fluid be carried out prehybridization.
Solid-phase hybridization between RNA of the present invention and the miRNA chip carries out according to the classical way of this area, and the general personnel in this area determine relevant damping fluid, probe and the optimum condition of concentration of specimens, prehybridization temperature, hybridization temperature and time etc. easily according to experience.Perhaps also can be with reference to described in " molecular cloning experiment guide ".
Obtain according to information such as the position of marking signal on the miRNA chip, intensity then and treat measurement information.If amplified production fluorophor mark, also can be directly obtain and treat measurement information with fluorescence detection device (as laser confocal scanning instrument Scanarray3000 etc.).
The purposes of miRNA chip
MiRNA of the present invention can be used to detect the express spectra of miRNA; Or be used to detect the regulative mode of miRNA for plant-growth or growth.
By miRNA chip of the present invention, can analyze these miRNA expression of gene patterns, can understand in the plant adjusting situation that corresponding gene pairs is coerced etc. (such as arid, high salt etc.); Can design corresponding strategy, regulate and modified target dna, improve adaptive faculty, perhaps form genetic modification crop with better economical character to environment such as coercing.
MiRNA chip of the present invention can be applicable to the molecular screening of crop breeding.According to the results of hybridization of miRNA chip, screening has the plant of certain expression pattern targetedly, and seed selection becomes to stablize strain.Improve the efficiency of selection of breeding.
Detection kit
The present invention also provides a kind of test kit, contains chip of the present invention in the described test kit.Described test kit can be used for detecting the express spectra of miRNA; Or be used to detect the regulative mode of miRNA for plant-growth or growth.
Preferred, also contain the marker that is useful on the labeled rna sample in the described test kit, and with the corresponding substrate of described marker.
In addition, also can comprise required all ingredients such as being used to extract RNA, PCR, hybridization, colour developing in the described test kit, include but not limited to: extract, amplification liquid, hybridization solution, enzyme, contrast liquid, colour developing liquid, washing lotion, antibody etc.
In addition, also can comprise working instructions and/or chip image analysis software in the described test kit.Described chip image analysis software is such as the BaiO ArrayDoctor 2.0 of BaiO company, the Arraypro 4.0 of MediaCybernetics company, by the dChip (DNA-Chip Analyzer) of joint developments such as Harvard University biological the Cheng Li of department of statistic, Wing Wong, by the microarray analysis software package MultiExperiment Viewer (MeV v3.1) of TIGR exploitation.
Major advantage of the present invention is:
To from plant, separate the new miRNA of a class that obtains and be prepared into chip, thereby provide fast way for the regulating effect of plant for the express spectra of research miRNA and miRNA.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, people such as Sambrook for example, molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.
Unless otherwise defined, the same meaning that employed all specialties and scientific words and one skilled in the art are familiar with in the literary composition.In addition, any method similar or impartial to described content and material all can be applicable among the present invention.The usefulness that preferable implementation method described in the literary composition and material only present a demonstration.
The acquisition of embodiment 1miRNA
1.RNA extracting
Select for use and spend No. 11 in the japonica rice variety, whole life was finished in cultivation in (photoperiod is: 28 ℃ of illumination 12 hours and 22 ℃ of dark 12 hours) in the controlled environment chamber in three months.
Bloomed in after planting 60 days, respectively at back 3 days, 6 days, 9 days, the 12 days rice paddy seeds of gathering of blooming.After grinding in the seed material liquid nitrogen, use the TRIZOL test kit, the specification sheets that carries according to the TRIZOL test kit extracts total RNA.
2.small the acquisition of RNA sequence and screening
Reclaim the small RNA of 18～26nt with 20%PAGE glue, after adopting ordinary method that small RNA is cloned into sequencing vector Tag vector pMBS I (Solexa company), obtain the Signature sequence with the order-checking of MPSS technology, mask and to be the sequence part in joint source, method therefor is: the Signature sequence and the sequencing vector sequence of total length are compared, sequence 3 ' end and carrier mate fully thinks the carrier sequence, and the sequence filter of credibility lower (as the signal that checks order very low etc.) is fallen.
3.miRNA evaluation
(NCBI navigates to small RNA Signature sequence on the rice chromosome www.ncbi.nlm.nih.gov) that (the TIGR annotation information, www.tigr.org), rejecting not have the sequence of mating on genome with blast program.
(the TIGR annotation information www.tigr.org) is reference, removes rRNA, tRNA, snRNA and snoRNA etc. with the Rfam database.
In remaining small RNA sequence, in order to differentiate miRNA wherein, the inventor chooses the precursor sequence that comprises described small RNA with the window (window) of 400bp on genome, (www.tbi.univie.ac.at) predicts its secondary structure with RNAfold software.The small RNA that satisfies following standard simultaneously is accredited as miRNA:
1. miRNA is positioned on a certain arm of front body structure (5 ' or 3 '), and the front body structure free energy is minimum;
2. be that the mispairing number must not be greater than 7 among the 25nt at center with miRNA;
3. there is not big ring texture on the stem of front body structure.
4. result
By aforementioned screening, obtain 137 new ripe miRNA, shown in SEQ ID NO:1-SEQ ID NO:137 (table 1).The sequence of the precursor miRNA of these miRNA is shown in SEQ ID NO:138-SEQ ID NO:274 (table 2).
Table 1
Table 2
The characteristics of each sequence are to form loop-stem structure in the table 2, and each sophisticated miRNA is from a wherein side of the stem of corresponding loop-stem structure in the table 2 in the table 1.And the precursor of the miRNA in the table 2 and each miRNA in the table 1 are one to one, and corresponding relation is as follows:
The preparation of embodiment 2miRNA chip
1. the design of probe is with synthetic
The miRNA sequence (SEQ ID NO:1-SEQ ID NO:137) that table 1 is provided converts reverse complementary sequence to, adds the catenation sequence of 10-20nt at the sequence two ends according to features such as the GC that produces sequence compare; Core sequence difference, catenation sequence are also different.Catenation sequence can be at random, but the probe that catenation sequence and core sequence form meets the following conditions:
1) in the probe sequence, is no more than 50% of sequence sum with the quantity of a kind of Nucleotide (A, C, G, T).
2) quantity of any successive A, T or C, G can not surpass 25% of sequence sum.
3) G, C content account for the 40%-60% of sequence sum.
4) probe sequence can not be from hybridization, and promptly complementary segmental length can not surpass 30% of probe length in the probe sequence.
For the synthetic probe stably is combined on the slide, adopt conventional method to carry out glycosyl modified at 5 ' end of synthetic back probe.
2.miRNA the some system of chip
Earlier the surface of slide is carried out alkylation and modify, to improve binding ability.Adopt conventional chip point sample method to carry out point sample, in order to detect the repeatability of cross experiment, each probe is 3-6 hybridization point of point on slide.
The application of embodiment 3miRNA chip
1.miRNA the hybridization of chip
1) the Trizol single stage method is extracted total RNA of tissue, concentrates RNA by the isopropanol precipitating method, and is quantitative with spectrophotometer, the quality of the total RNA of denaturing formaldehyde gel electrophoresis quality inspection.Get the total RNA of 50-100 μ g, use the small fragment RNA of Ambion ' s miRNA Isolation Kit (Cat#.1560) separation length about 10-100bp.Utilize conventional marking method (T4RNA ligase enzyme labelling method) to carry out mark, labeled substrate is cy3, and then with the dehydrated alcohol precipitation, is used for and chip hybridization after drying up.
2) RNA is dissolved in (15% methane amide in the 16 μ L hybridization solutions; 0.2%SDS; 3 * SSC; 50 * Denhardt ' s), spend the night in 42 ℃ of hybridization.After hybridization finishes, contain 0.2%SDS earlier about 42 ℃, wash 4min in the liquid of 2 * SSC, then room temperature is washed 4min in 0.2 * SSC liquid.
After drying, slide promptly can be used for scanning.
Fig. 2 has shown the part of the results of hybridization after the scanning.
2. the analysis of results of hybridization
Adopt conventional chip signal to read instrument, the picture signal that scans is converted to numerary signal, and carries out signal correction, remove the signal value of bad point and weakness.
Use the related software analysis, the difference condition of miRNA genetic expression.Described chip analysis software is such as microarray analysis software package MultiExperiment Viewer (MeVv3.1) that dChip (DNA-ChipAnalyzer) that is developed jointly by Harvard University biological the Cheng Li of department of statistic, Wing Wong etc. and TIGR release.Method that analyze to express difference condition is described below: according to different tissues chip data is divided into groups, the data between every group are with the method for ANOVA check, and statistical study is the difference condition between on the same group not.The P value is set at less than 0.05.
Spend the miRNA express spectra in 11 the multiple tissue in embodiment 4 paddy rice
1.miRNA the acquisition of expression data
Prepare to spend 11 multiple tissue (comprising: seed, leaf, root, endosperm, embryo) in the paddy rice, adopt ordinary method to separate small fragment RNA respectively, adopt the chip of embodiment 2 preparations, carry out chip hybridization, obtain the expression of results of all miRNA in each tissue according to the method among the embodiment 3.Various tissues need biological the repetition and the technology repetition.
2.miRNA the establishment of express spectra
Use related software, all data normalizations are handled.With the equal method normalization of all signal medians of whole chip.Calculate the mean value of all miRNA signals of homologue.
MeV v3.1 with TIGR shows the expression of all miRNA in different tissues, is compiled into the miRNA express spectra.The result as shown in Figure 1.
Show that by the expression map result expression of most of miRNA has tissue specificity, concentrate in one to two tissue to express that miRNA is in the special functionating of organizing in prompting.In addition, most of miRNA expresses very low in endosperm, and is similar with the expression pattern of gene mRNA.Therefore can change the expression pattern of target gene to a great extent by influence the expression pattern of miRNA, and play the purpose of change crop economical character etc.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. a miRNA chip is characterized in that, described miRNA chip comprises:
Solid phase carrier; And
Be fixed on the oligonucleotide probe on the described solid phase carrier in order, described oligonucleotide probe is specifically corresponding to the part or all of sequence shown in the SEQ ID NO:1-SEQ ID NO:137.
2. miRNA chip as claimed in claim 1 is characterized in that, described oligonucleotide probe contains:
Complementary land; And/or
The joining region that links to each other with solid phase carrier.
3. miRNA chip as claimed in claim 2 is characterized in that, the complementary land of described oligonucleotide probe is: be selected from the sequence complementary sequence shown in the SEQ ID NO:1-SEQ ID NO:137.
4. miRNA chip as claimed in claim 1 is characterized in that, described oligonucleotide probe is by 20-80 based composition.
5. the purposes of the described miRNA chip of claim 1 is characterized in that, is used for:
Detect the express spectra of miRNA; Or
Detect the regulative mode of miRNA for plant-growth or growth.
6. a method that detects miRNA express spectra in the plant is characterized in that, comprises step:
(1) provides separation from the RNA of plant sample, on described RNA, marker is set;
(2) RNA with (1) contacts with the described chip of claim 1, makes the oligonucleotide probe generation hybridization on described RNA and the solid phase carrier, thereby forms " oligonucleotide probe-RNA " binary complex on solid phase carrier; With
(3) detect the marker of the binary complex of (2) formation, thereby determine the express spectra of corresponding miRNA in the plant.
7. method as claimed in claim 6 is characterized in that, in step (1), also comprises step before marker is set: enrichment small fragment RNA from the RNA sample.
8. method as claimed in claim 7 is characterized in that, the length of described small fragment RNA is 10-100bp.
9. method as claimed in claim 6 is characterized in that, described marker is selected from: fluorescein or derivatives thereof, digoxin, vitamin H, alkaline phosphatase, or horseradish peroxidase.
10. a test kit is characterized in that, contains the described miRNA chip of claim 1 in the described test kit.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
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| CNA2007100365763A CN101225433A (en) | 2007-01-18 | 2007-01-18 | Plant miRNA chip and uses thereof |
| CN2008800026220A CN101605901B (en) | 2007-01-18 | 2008-01-18 | miRNA of plant, chip and the uses thereof |
| PCT/CN2008/070140 WO2008089695A1 (en) | 2007-01-18 | 2008-01-18 | Mirnas of plant, its chip and the uses thereof |
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| CNA2007100365763A CN101225433A (en) | 2007-01-18 | 2007-01-18 | Plant miRNA chip and uses thereof |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102899418A (en) * | 2012-11-09 | 2013-01-30 | 中国科学院上海应用物理研究所 | Electrochemical miRNA (micro Ribose Nucleic Acid) detection method based on DNA (Deoxyribose Nucleic Acid) three-dimensional nano structure probe |
| CN110904109A (en) * | 2019-12-16 | 2020-03-24 | 河南农业大学 | miR1866 gene for controlling rice seed germination, overexpression vector, gRNA expression vector, preparation method and application thereof |
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2007
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102899418A (en) * | 2012-11-09 | 2013-01-30 | 中国科学院上海应用物理研究所 | Electrochemical miRNA (micro Ribose Nucleic Acid) detection method based on DNA (Deoxyribose Nucleic Acid) three-dimensional nano structure probe |
| CN110904109A (en) * | 2019-12-16 | 2020-03-24 | 河南农业大学 | miR1866 gene for controlling rice seed germination, overexpression vector, gRNA expression vector, preparation method and application thereof |
| CN110904109B (en) * | 2019-12-16 | 2023-04-11 | 河南农业大学 | miR1866 gene for controlling rice seed germination, overexpression vector, gRNA expression vector, preparation method and application thereof |
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