CN102250899B - MiRNA-GramiRn22 from cotton and applications thereof - Google Patents
MiRNA-GramiRn22 from cotton and applications thereof Download PDFInfo
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Abstract
The invention discloses miRNA-GramiRn22 from cotton and applications thereof. The miRNA-GramiRn22 protected by the invention is RNA represented by SEQ ID NO:1 in a sequence table, and a precursor of the miRNA-GramiRn22 (pre-gramiRn22) is RNA represented by SEQ ID NO:2 in the sequence table. Applications of the RNA represented by SEQ ID NO:1 and/or SEQ ID NO:2 in inhibiting pre-mRNA splicing factor gene expression and/or promoting mRNA degradation of the pre-mRNA splicing factor gene are protected, the pre-mRNA splicing factor is represented by a SEQ ID NO:3 in the sequence table, and the pre-mRNA splicing factor gene is represented by SEQ ID NO:4 in the sequence table. Plants with important phenotypes in aspects of adversity stress tolerance and growing development may be obtained through adopting the miRNA-GramiRn22, so the miRNA-GramiRn22 has important biological significances and latent application values, provides precious gene resources for cotton quality breeding (stress resistant cotton cultivation for example), brings certain research values and social benefits, and finally can be used in practical production.
Description
Technical field
The present invention relates to a kind of miRNA-GramiRn22 and application thereof that derives from cotton.
Background technology
MiRNA (microRNA, Microrna) is the endogenous strand non-coding microRNA that a class length is about 20-24nt, in organism, extensively exists.Large quantity research shows in recent years, and miRNA can regulate and control in organism to be permitted polygenic expression, at growth regulation, cell proliferation, apoptosis, resist all many-sides such as environment-stress and play a significant role.Mirnas of plant is mainly by cutting target gene mRNA, or the translation of inhibition said target mrna, and carry out regulating plant individual growth and grow and affect its physiological process, be a kind of new gene regulating pattern, there is important Research Significance.
Cotton is one of most important cash crop in the world, and cotton fiber is also the good model of the unicellular elongation of research simultaneously.With Arabidopis thaliana, paddy rice isotype biophase ratio, the cotton miRNA quantity having been found that is at present very few, therefore pass through high throughput sequencing technologies, be expected to excavate more cotton miRNA, this forming process, constructional feature and functional mechanism for overall understanding cotton and even whole Mirnas of plant has realistic meaning.In addition, in cotton, miRNA may be at many physiological processs (as the initial of fiber and elongation etc.) performance critical function, but the biological function research about miRNA in cotton is very few, therefore, by paying close attention to the miRNA in specific developmental stage, particular organization, be expected to illustrate the physiological process that cotton miRNA participates in, and the effect of concrete performance in this process.
China is main in the world Cotton Production and country of consumption, and cotton has very important status for China.Both at home and abroad except utilizing conventional breeding means, progressively using genetic engineering technique to carry out genetic improvement to cotton quality (output, pest-resistant etc.) has become a kind of trend.Because miRNA has regulating and controlling effect widely to plant, probably become one of primary study gene of genetic modification of plants, therefore in the urgent need to fully excavate and develop the new miRNA gene that belongs to national intellecture property by large scale sequencing method, thereby lay the foundation for later stage orderly improvement and the cotton variety of cultivating high-quality proterties.
Summary of the invention
The object of this invention is to provide a kind of miRNA-GramiRn22 and application thereof that derives from cotton.
The sequence following (5 ' → 3 ') of the miRNA-GramiRn22 of the present invention's protection:
UAUGAAACUGUGAUUCCACGUCAU (sequence 1 of sequence table).
MiRNA-GramiRn22 precursor (pre-GramiRn22) sequence following (5 ' → 3 ') of the present invention's protection:
AAUGGACUUGUAUGAAACUGUGAUUCCACGUCAUUUUUAUCCCUUUCCUUUAGGAGAAUGACAAAUCAGAUUUUUCCTACU-78nt-AGGAACAACGUGGAAUCACAAUUUCAUACAAUCCUUU。(sequence 2 of sequence table).
The present invention also protects RNA shown in the sequence 2 of RNA shown in the sequence 1 of sequence table and/or sequence table suppressing the application of mRNA precursor splicing factor (TC255234) in expressing; Described mRNA precursor splicing factor is as shown in the sequence 3 of sequence table. as described in mRNA precursor splicing factor gene specifically can be as shown in the sequence of sequence table 4 or the sequence 4 of sequence table from shown in the 12nd to 590 Nucleotide of 5 ' end.
The present invention also protects the application of RNA in the mRNA degraded that promotes mRNA precursor splicing factor gene (TC255234) shown in the sequence 2 of RNA shown in the sequence 1 of sequence table and/or sequence table; Described mRNA precursor splicing factor is as shown in the sequence 3 of sequence table. as described in mRNA precursor splicing factor gene specifically can be as shown in the sequence of sequence table 4 or the sequence 4 of sequence table from shown in the 12nd to 590 Nucleotide of 5 ' end.
Solexa has overcome the shortcoming of conventional miRNA clone technology, has advantages of highly sensitively, can detect a minimum small RNA molecular, and accuracy is high, and the small RNA molecular base error rate detecting is extremely low.This technology also has high-throughput, large-scale feature simultaneously, and the order-checking amount in two libraries is up to 5,000,000 more than little RNA sequence.Based on this up-to-date order-checking means, the new miRNA of specific developmental stage particular organization specifically expressing in cotton will be expected to identify.
The present invention adopts advanced in the world Solexa high throughput sequencing technologies in conjunction with various biological means such as bioinformatic analysis, Northern hybridization, 5 ' RACE, identify miRNA-GramiRn22 from genomic level first, and confirm that the target gene of GramiRn22 is mRNA precursor splicing factor gene, participated in the regulation and control of the initial and Ovule Development of cotton fiber.This all provides valuable genetic resources by the quality breeding for cotton (as improved production of cotton fibers), brings certain researching value and social benefit, and finally for actual production.MiRNA wide participation provided by the invention the adjusting of the multiple vital movement of cotton, there is important biological significance and potential using value.
Accompanying drawing explanation
Fig. 1 is the schema that separates and identify new miRNA in little RNA sequencing data.
Fig. 2 is the precursor secondary structure figure of miRNA-GramiRn22; Black line part is indicated the position at ripe miRNA place.
Fig. 3 is the expression to miRNA-GramiRn22 in bloom latter 3 days wild-types and mutant cotton ovule in first 3 days of blooming of Northern hybridization check.
Fig. 4 is target gene 5 ' the RACE checking of miRNA-GramiRn22; The arrow of sequence top represents to occur the site of cutting, and clone's number and the total ratio of clone of cutting occurs at numeric representation this point of contact place.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment, results averaged for three times.
In following examples, cotton variety used comprises upland cotton Xuzhou 142 (Gossypium hirsutum cv.Xuzhou 142, below, referred to as wild-type) and without Mutants of Fiber (being designated hereinafter simply as mutant), cotton seeds derives from National Cotton storehouse in mid-term, wherein wild-type storehouse number is 110599, and mutant library number is 140142.
Cotton was planted in field the last ten-days period in April, routine work, and petal bagging the day before yesterday of blooming, prevents that pollen transmission from causing cross-pollination, the same day of blooming is except bag, and listing mark.Collect respectively bloom first 3 days (3), the cotton boll of bloom first 1 day (1), bloom the same day (0), bloom latter 1 day (1), bloom latter 3 days (3), cut open and get ovule, rapidly frozen in liquid nitrogen, be stored in-80 ℃ for subsequent use.
The discovery of embodiment 2, miRNA-GramiRn22
1, RNA extracts
The cotton ovule sample that embodiment 1 is obtained grinds in liquid nitrogen, adds PVP (by 1/5 mass ratio) to prevent phenols oxidation in process of lapping, uses PureLink
tMplant RNA Reagent (Invitrogen) extracts total RNA, operation steps following (take 0.1g material as example, corresponding reagent amount can be adjusted in proportion according to quantity of material):
1. the ovule powder of milled joins in 1ml Extraction buffer, adds 20 μ l beta-mercaptoethanols, mixes and is placed on room temperature 10-15 minute.4 centrifugal 5 minutes of 12000 revs/min of degree, go to new centrifuge tube by supernatant, add 100 μ l5M NaCl, add 300 μ l chloroforms after mixing, and fully mix, and 4 centrifugal 10 minutes of 12000 revs/min of degree, go to new centrifuge tube by supernatant;
2. the solution producing is used to chloroform, phenol, phenol successively: chloroform (1: 1), chloroform extracting, supernatant liquor after four extractings proceeds to new centrifuge tube, add 100 μ l polysaccharide removers (ocean bio tech ltd of Beijing CHMC), after mixing, add 200 μ l chloroforms, fully mix, 4 centrifugal 10 minutes of 12000 revs/min of degree, go to new centrifuge tube by supernatant;
3. add isopyknic Virahol, mix postposition-20 and spend more than 1 hour, 4 centrifugal 10 minutes of 12000 revs/min of degree, abandon supernatant, and 75% washing with alcohol for the centrifugal precipitation obtaining, is dissolved in after drying in appropriate DEPC water.
Use Ultrospec 3000 type ultraviolet spectrophotometers (Amersham Biosciences) to measure the RNA extracting at 260nm (OD
260) and 280nm (OD
280) absorbance of wavelength to be to determine purity and the concentration of RNA.Up-to-standard RNA concentration should be more than 1 μ g/ μ l, OD
260/ OD
280ratio between 1.8-2.0, and clear through electrophoresis detection band, pollute without obvious degradation and DNA.
2, the structure in little RNA library
Total cotton ovule qualified quality test RNA is used for building little RNA library.The RNA of the ovule sample extraction of 5 different times respectively gets 10 μ g balanced mix, and 50 μ g are used for building little RNA library altogether.The structure in little RNA library carries out according to Illumina Sample Preparation Protocol library constructing method, the library building adopts Solexa high-flux sequence (Huada Gene Research Center, Beijing), obtains the little RNA sequence of high-quality 18-30nt.
3, the evaluation of new miRNA in little RNA library
Successful methods (the Jones-Rhoades M W of foreign literature to high-flux sequence data analysis before reference, Bartel D P.Computational identification of plant miRNAs and their targets, including a stress-induced miRNA.Mol.Cell, 2004,14:787-799.), set up the cotton miRNA (analysis process as shown in Figure 1) that a set of computer analysis method is used in discovery and evaluation sequencing data.1. the original series in two little RNA libraries that obtain is removed to 3 ' joint by computer approach, and filter out the sequence of sequence length below 18nt, obtain so-called " totally " sequence library, 2. the miRNA mature sequence of announcing in the miRNA database miRBase (http://microrna.sanger.ac.uk/sequences/) of the sequence of " totally " and internal authority is carried out to BLAST, thereby find which sequence comes from known miRNA, the sequence that known miRNA exceedes 2 mispairing enters next step analysis again, 4. by got rid of for the sequence of conservative miRNA again with known organelle gene database, and other non-coding RNA database, comprise Arabidopis thaliana Mitochondrial DNA (www.arabidopsis.org), rape Mitochondrial DNA (NCBI), upland cotton plastid DNA (NCBI), plant snRNA and snoRNA (NCBI), upland cotton and Arabidopis thaliana rRNA (NCBI), and tRNA (http://gtrnadb.ucsc.edu) mates, thereby find which sequence comes from organoid RNA and other non-coding RNA, filter out these sequences, in remaining sequence, may contain new miRNA, be called potential miRNA sequence storehouse, 5. the sequence in potential miRNA sequence storehouse and existing cotton database are carried out to BLAST, database comprise cotton EST (
http://compbio.dfci.harvard.edu), the genome sequence of cotton GSS (NCBI) and existing part Lei Mengdeshi cotton (Gossypium raimondii).Sequence corresponding in the cotton database finding is carried out to next step analysis.6. use miRNA front body structure forecasting software mireap_0.2 (http://sourceforge.net/projects/mireap), to there being the sequence that little RNA is corresponding to carry out secondary structure analysis in the cotton database obtaining, if the good loop-stem structure that can form similar miRNA precursor (pre-miRNA) this sequence can think candidate's new miRNA, 7. candidate's new miRNA is proceeded to screening, investigate the little RNA distribution characteristics of the loop-stem structure precursor at candidate's new miRNA sequence place, if be mainly distributed in candidate's new miRNA region and corresponding miRNA* region, think that this candidate's new miRNA sequence height is credible, real miRNA sequence (Meyers B C, Axtell M J, Bartel B, Bartel D P, Baulcombe D, Bowman J L, Cao X, Carington J C, Chen X, Green P J, et al.Criteria for annotation of plant microRNAs.Plant Cell, 2008, 20:3186-3190.).
Following (5 ' → 3 '): the UAUGAAACUGUGAUUCCACGUCAU of sequence of miRNA-GramiRn22.
Order-checking number of times (the Q in the little RNA of wild-type library
wT) be 43, the little RNA of mutant library (Q
mU) order-checking number of times be 58.
The secondary loop-stem structure of miRNA precursor (pre-miRNA) sequence is one of outstanding feature of miRNA gene, is also all impassable important rule of all miRNA authentication methods.
GramiRn22 precursor (pre-gramiRn22) sequence following (5 ' → 3 '):
AAUGGACUUGUAUGAAACUGUGAUUCCACGUCAUUUUUAUCCCUUUCCUUUAGGAG AAUGACAAAUCAGAUUUUUCCTACU-78nt-AGGAACAACGUGGAAUCACAAUUUC AUACAAUCCUUU (sequence 2 of sequence table).
Pre-gramiRn22 can form good loop-stem structure, and ripe miRNA produces from the stem of miRNA precursor, meets the constitutional features (seeing Fig. 2) of miRNA precursor completely.
For further detect miRNA-GramiRn22 before blooming 3 days to 3 days expression patterns in cotton ovule after blooming, adopt its expression of miRNA Northern hybridization check.
1, prepare probe
Detect the sequence of the probe (5 ' → 3 ') of miRNA-GramiRn22: ATGACGTGGAATCACAGTTTCATA.
Adopt T4 polynueleotide kinase (New England Biolabs) to carry out isotropic substance (γ-32P ATP) mark to above-mentioned sequence terminal phosphate group, obtain probe, with Microspin G-25 post (GE Healthcare) purifying probe, remove unlabelled isotropic substance, the probe after purifying is for Northern hybridization.
2, miRNA Northern hybridization
In Northern hybridization, each swimming lane applied sample amount is the total RNA of 30 μ g, and U6RNA, as internal reference, detects on same film with miRNA.The probe sequence of U6 gene is: TGTATCGTTCCAATTTTATCGGATGT.
(1) extract respectively total RNA of each ovule sample that embodiment 1 obtains.
(2) miRNA Northern hybridization
1. the total RNA that gets 30 μ g amounts, adds isopyknic 2 × RNA sample-loading buffer (95% methane amide, 18mM EDTA, 0.1% tetrabromophenol sulfonphthalein and 0.1% dimethylbenzene green grass or young crops), mixes rear 95 ℃ of sex change 5min, obtains RNA sample.
2. RNA sample separates with 15% urea-denatured polyacrylamide gel electrophoresis, and electricity consumption transfer device (BIO-RAD) is transferred on Hybond N+ nylon membrane (Amersham Biosciences) subsequently.
3. shift and have the nylon membrane of RNA sample through the of short duration rinsing of 6 × SSC solution, UV-crosslinked (Stratagene) 5min, then 80 ℃ of baking 2h, be completely fixed on nylon membrane RNA.
4. the nylon membrane that has shifted RNA is put into hybrid pipe, add 5ml ULTRAhyb-Oligo hybridization solution (Ambion) 42 ℃ of prehybridization 2h in hybrid heater, then add probe, mix, 42 ℃ of hybridization are spent the night.
5. after hybridization finishes, carefully pour out hybridization solution, add the 2 × SSC solution containing 0.5%SDS, 42 ℃ are washed three times, each 10min.
6. after washing film and finishing, film is wrapped up to smooth being pressed under X-ray, additional intensifying screen ,-70 ℃ of exposures 2 weeks with preservative film.
7. after end exposure, rinse X-ray, carry out densitometric scan, take the hybridization signal of control group as 1, calculate the relative intensity of each treatment group hybridization signal.
Northern results of hybridization is shown in Fig. 3.In wild-type (WT), miRNA-GramiRn22 before blooming 3 days to after blooming 3 days, expression amount presents ascendant trend substantially.And in mutant (MU), the expression amount of miRNA-GramiRn22 is on a declining curve, imply that GramiRn22 may play an important role in cotton fiber initiating process.
Target gene prediction and the checking of embodiment 4, miRNA
Due to Mirnas of plant and the intimate complete complementary of target gene mRNA, therefore can predict by bioinformatics method the target gene of miRNA-GramiRn22.Adopt online software psRNATarget (http://plantgrn.noble.org/psRNATarget/) in cotton est database CGI10, to search out cDNA or the gene that can be close to miRNA sequence complete complementary, be the target of miRNA; Parameter is set to: psRNATarget program parameter is default setting, and the function of target is by NCBI (http://www.ncbi.nlm.nih.gov/) homology search, and the known function gene the highest with homology annotates.
Predict the outcome in table 1.
Target gene and the function of table 1miRNA-GramiRn22
The target gene of miRNA-GramiRn22 is that mRNA precursor splicing factor (pre-mRNA splicing factor) gene TC255234 (is shown in the sequence 4 of sequence table, the protein of its coding is shown in the sequence 3 of sequence table), participate in the processing of a series of important gene mRNA precursors.In Arabidopis thaliana, this gene (atSRp30) is a kind of mRNA precursor splicing factor, the transfer-gen plant of this gene of overexpression is compared with wild-type plant, there were significant differences for its blade trichome development, the growth that this gene pairs blade table fur is described has important regulating and controlling effect (Sergiy Lopato, Maria Kalyna, Silke Dorner, Ryuji Kobayashi, Adrian R.Krainer, and Andrea Barta.atSRp30, one of two SF2/ASF-like proteins from Arabidopsis thaliana, regulates splicing of specific plant genes.Genes & Development, 1999, 13:987-1001.).This gene is also brought into play important regulating and controlling effect in cotton fiber development.
MiRNA-GramiRn22 verifies (Jones-Rhoades M W to the cutting of target gene TC255234 (seeing the sequence 4 of sequence table) mRNA by 5 ' RACE method, Bartel D P.Computational identification of plant miRNAs and their targets, including a stress-induced miRNA.Mol.Cell, 2004,14:787-799.).After target gene mRNA is cut by miRNA, its 3 ' comparatively stable cleaved products 5 ' terminal nucleotide phosphate group exposes, and uses T4RNA ligase enzyme to connect upper 5 ' RACE special joint at this cleaved products 5 ' end; Synthesize cDNA by reverse transcription reaction; By the special nido outer primer of target gene and test kit with nido outer primer carry out first round PCR, the nido inner primer that target gene is special and test kit with nido inner primer carry out second and take turns PCR; After the PCR product that 5 ' RACE is obtained is connected to pMD 19-T carrier, check order, just can know accurate target gene mRNA cleavage site.Primer in above-mentioned 5 ' RACE method is for target gene TC255234 (being shown in the sequence 4 of sequence table).
1, extract respectively total RNA of each ovule sample that embodiment 1 obtains.
2, be cDNA by total RNA reverse transcription of step 1; Carry out 5 ' RACE by Firstchoice RLM-RACE test kit (Ambion) operation instructions, with cDNA for template pass through the special nido outer primer of target gene (5 '-ATCCTCCCGATCTCCTTCCACAGT-3 ') and test kit with nido outer primer carry out first round PCR, with first round PCR product for template pass through the special nido inner primer of target gene (5 '-TTGCTGAACTCCAGGAAATGTGTCGG-3 ') and test kit with nido inner primer carry out second and take turns PCR.
3, by second taking turns PCR product and carry out agarose electrophoresis in step 2, reclaim the specific band of 150bp left and right.
4, specific band step 3 being reclaimed is received pMD 19-T carrier (TaKaRa), and transforms bacillus coli DH 5 alpha (purchased from American I nvitrogen company, catalog number is 18258-012).
5, choose mono-clonal order-checking, determine the cleavage site of target gene mRNA according to sequencing result.
The cleavage site that sequencing result shows is shown in Fig. 4.There is cutting in the region complementary with it in the target gene of miRNA-GramiRn22, this result is strong has proved that TC255234 is the target gene of real regulation and control in miRNA-GramiRn22 body really.
Claims (6)
1. the RNA shown in the sequence 1 of sequence table.
2. the RNA shown in the sequence 2 of sequence table.
3. RNA shown in RNA shown in sequence 1 and/or sequence 2 is in the application suppressing in the genetic expression of mRNA precursor splicing factor; Described mRNA precursor splicing factor is as shown in the sequence 3 of sequence table.
4. application as claimed in claim 3, is characterized in that: described mRNA precursor splicing factor gene as shown in the sequence 4 of sequence table or the sequence 4 of sequence table from shown in the 12nd to 590 Nucleotide of 5 ' end.
5. the application of RNA shown in RNA shown in sequence 1 and/or sequence 2 in the mRNA degraded that promotes mRNA precursor splicing factor gene; Described mRNA precursor splicing factor is as shown in the sequence 3 of sequence table.
6. application as claimed in claim 5, is characterized in that: described mRNA precursor splicing factor gene as shown in the sequence 4 of sequence table or the sequence 4 of sequence table from shown in the 12nd to 590 Nucleotide of 5 ' end.
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| Cesar Liave 等.Cleavage of Scarecrow-like mRNA Targets Directed by a Class of Arabidopsis miRNA.《SCIENCE》.2002,第297卷(第20期),2053-2056. |
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