CN109536496A - The precursor-gene of shiny-leaved yellowhorn miR169a and and its application in flowering of plant in advance - Google Patents
The precursor-gene of shiny-leaved yellowhorn miR169a and and its application in flowering of plant in advance Download PDFInfo
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Abstract
The invention discloses the precursor-genes of shiny-leaved yellowhorn miR169a a kind of, the sequence of the precursor-gene is as follows: (a) nucleotide sequence as shown in SEQ ID NO:1, or (b) at least has 70% with nucleotide sequence shown in SEQ ID NO:1, at least with 75%, at least with 80%, at least with 85%, at least with 90%, at least with 95%, at least with 96%, at least with 97%, at least with 98% or at least has the nucleotide sequence of 99% homology and the clipped shiny-leaved yellowhorn miR169a of generation afterwards.The present invention also provides application of the precursor-gene of shiny-leaved yellowhorn miR169a in flowering of plant in advance.Using the precursor-gene of shiny-leaved yellowhorn miR169a, it is expected to artificially shift to an earlier date flowering of plant.
Description
Technical field
The present invention relates to the precursor-genes of shiny-leaved yellowhorn miR169a a kind of, and disclose the precursor base of shiny-leaved yellowhorn miR169a
Because of the application in flowering of plant in advance.
Background technique
MicroRNA (miRNA) is that one kind is widely present in animals and plants, the miRNA precursor containing loop-stem structure, is passed through
The small RNA molecular (18-25 nucleotide) of a kind of non-coding after Dicer processing.MiRNA by transcriptional level or turn
Level inhibits its translation or cutting target gene to carry out controlling gene expression the target gene on genome after record, in gene expression
Play its target gene of negative regulation.The entire flower development mistake that miRNA is formed in plant from floral induction to floral organ characteristic attribute
Cheng Jun plays key effect.
MiR169 is a kind of miRNA being widely present in monocotyledon and dicotyledon.In arabidopsis,
The target gene of miR169 is NF-Y family member, regulates and controls stress response, while miR169 by inhibiting NF-YA genetic transcription
It is overexpressed and promotes arabidopsis Blooming.However, shiny-leaved yellowhorn miR169a function is on the knees of the gods.
Summary of the invention
The object of the present invention is to provide the precursor-gene of shiny-leaved yellowhorn miR169a a kind of, the sequences of the precursor-gene
It is as follows:
(a) nucleotide sequence as shown in SEQ ID NO:1, or
(b) at least have 70% with nucleotide sequence shown in SEQ ID NO:1, at least have 75%, at least have
80%, at least have 97% with 85%, at least with 90%, at least with 95%, at least with 96%, at least, at least have
Have 98% or at least with 99% homology and the clipped rear nucleotide sequence for generating shiny-leaved yellowhorn miR169a.
In one embodiment, the sequence of shiny-leaved yellowhorn miR169a is SEQ ID NO:2.
The present invention also provides the recombinant vector, transgenic cell line or the recombinant bacteriums that contain above-mentioned precursor-gene.Preferably,
The recombinant bacterium is the recombinant bacterium for obtaining above-mentioned precursor-gene insertion expression vector.
On the other hand, the present invention also provides the precursor-genes of above-mentioned shiny-leaved yellowhorn miR169a in flowering of plant in advance
Using the sequence of the precursor-gene of the shiny-leaved yellowhorn miR169a is as follows:
(a) nucleotide sequence as shown in SEQ ID NO:1, or
(b) at least have 70% with nucleotide sequence shown in SEQ ID NO:1, at least have 75%, at least have
80%, at least have 97% with 85%, at least with 90%, at least with 95%, at least with 96%, at least, at least have
Have 98% or at least with 99% homology and the clipped rear nucleotide sequence for generating shiny-leaved yellowhorn miR169a.
In one embodiment, the sequence of the precursor-gene of above-mentioned shiny-leaved yellowhorn miR169a is SEQ ID NO:1.
In one embodiment, the sequence of above-mentioned shiny-leaved yellowhorn miR169a is SEQ ID NO:2.
In one embodiment, above-mentioned plant is arabidopsis or tobacco.
In one embodiment, above-mentioned plant is arabidopsis.
In one embodiment, above-mentioned application further include: will be connected comprising the precursor-gene of the shiny-leaved yellowhorn miR169a
Onto carrier, by agrobacterium mediation converted to arabidopsis, screening, culture and transgenic line is obtained.
It is an advantage of the current invention that the florescence of regulation plant is expected to, so that plant opens in advance using shiny-leaved yellowhorn miR169a
Flower, it is solid.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
In order to make the objectives, technical solutions, and advantages of the present invention clearer, combined with specific embodiments below to this
Invention is described in further detail.It should be appreciated that described herein, specific examples are only used to explain the present invention, is not used to
Limit the present invention.
1. material
1.1 experimental material
Experimental material is shiny-leaved yellowhorn bud, is derived from Liaoning Zhangwu shiny-leaved yellowhorn of Forestry Institue, Chinese Inst.of Forestry Sciences
2 years raw saplings of study base, kind are middle stone 4.Sample adopt it is lower after Liquid nitrogen storage immediately, be placed on -70 DEG C of refrigerators and save
For use.Arabidopsis wild type seeds use Columbia ecotype (Columbia-0).
1.2 experiment reagents and instrument
Archaeal dna polymerase, various restriction enzymes, T4 ligase, Marker, TRIzol, plasmid extraction kit and DNA
Plastic recovery kit reagent is purchased from Tiangeng biochemical technology Co., Ltd.PCR instrument is U.S. PE9700PCR instrument, superclean bench
Science and Technology Ltd. is really purified purchased from Suzhou.
The synthesis of 1.3 primers and sequencing
Primer synthesis and sequencing are completed by Shanghai Ying Jun Bioisystech Co., Ltd.
2. method
The extraction of 2.1 shiny-leaved yellowhorn total serum IgEs
The RNA (Total RNA) of shiny-leaved yellowhorn bud is extracted using improvement CTAB+ isopropanol precipitating method, steps are as follows:
(1) in 10mL centrifuge tube be added 65 DEG C of 3mL preheating CTAB Extraction buffers (10%CTAB, 10%PVP40,
1.0M Tris-HCl (pH 8.0), 5M NaCl, 0.6M EDTA (pH 8.0)), 200 μ L beta -mercaptoethanols are added；
(2) the bud 2g for taking -70 DEG C of preservations, is put into the mortar that liquid nitrogen is sufficiently pre-chilled, is fully ground in liquid nitrogen to hundred
Color powder；
(3) white powder is transferred to rapidly in the extracting solution of preheating, sufficiently oscillation mixes immediately, 65 DEG C of water-bath 30min, the phase
Between vibrate 3-4 times；
(4) isometric 25:24:1 (acid saturated phenol: chloroform: isoamyl alcohol), about 3ml are added in the mixture.Mixing is equal
14000rpm centrifugation 10min (room temperature) after even；
(5) supernatant is taken to be transferred in new 10ml centrifuge tube, be added isometric chloroform/isoamyl alcohol (24:1) turn upside down it is mixed
It is even, supernatant is taken after 12,000 4 DEG C of rpm centrifugation 10min；
(6) supernatant is transferred in 1.5mL centrifuge tube, is placed in -20 DEG C of refrigerator freezings after isometric isopropanol is added
30min.12000rpm4 DEG C of centrifugation 10min discards supernatant, and 65 DEG C of preheated SSTE400 μ L solution are added to Quan Rong in precipitating；
(7) 4 DEG C of 12000rpm are centrifuged 15min；
(8) supernatant is drawn, isometric isopropanol is added, is stored at room temperature 10min；
(9) 4 DEG C of 12000rpm are centrifuged 10min, discard supernatant, and the visible RNA of naked eyes is sunken to tube bottom；
(10) the 75% ethanol washing precipitating of 1mL pre-cooling is added, mild to vibrate, 8000rpm4 DEG C of centrifugation 5min is discarded
Clearly, and by precipitating it is dried in vacuo 7-10min；Appropriate DEPC water dissolution precipitating is added, -80 DEG C save backup.
2.2 gene cloning
After the RNA (Total RNA) of shiny-leaved yellowhorn is diluted with appropriate DEPC ddH2O, referring to PrimeScriptTM RT
The specification of Reagent Kit (Perfect Real Time) carries out the synthesis of the first chain of reverse transcription cDNA.Prepare following gene
Clone PCR system (20ul):
Response procedures are as follows: 95 DEG C, 3min；It 95 DEG C, 30sec, 58 DEG C, 30sec, 72 DEG C, 2min, recycles 37 times；72℃,
8min。
Gene cloning the primer (table 1).
The detection of 2.3 agarose gel electrophoresis is recycled with glue
By PCR reaction product carry out 1% agarose gel electrophoresis, electrophoretic buffer be TAE (40mM Tris- acetate,
1mM EDTA), nucleic acid fuel is EB.Ultraviolet light detection electrophoresis result simultaneously recycles PCR product.Utilize the DNA of Shanghai Sangon Biotech Company
Plastic recovery kit carries out glue recycling, the specific steps are as follows:
(1) target DNA fragment and other segments are separated as far as possible by agarose gel electrophoresis, with clean scalpel
Piece cuts the Ago-Gel block containing target DNA, is put into 1.5mL centrifuge tube.Each blob of viscose tries not to be more than
Otherwise it is incomplete will to will lead to colloidal sol by 400mg.In addition cutting glue process should be as fast as possible, to reduce DNA exposure in ultraviolet light
Time reduces the damage to DNA.
(2) according to the quality of blob of viscose and concentration, every 100mg agarose adds the ratio of 300~600 μ L that Buffer B2 is added.
(3) 55 DEG C of metal bath 10min are placed in, during which mix 2~3 times until blob of viscose dissolves completely.
(4) as target fragment < 500bp, the isopropanol of the Buffer B2 of 1/3 volume can be added, mix.If >=
500bp, then this step can be omitted.
(5) solution dissolved is all moved into adsorption column, 8,000xg centrifugation 30sec.The liquid in collecting pipe is outwelled,
And adsorption column is put into the same collecting pipe.
(6) 500 μ L Wash Solution, 9000xg are added and are centrifuged 30sec.The liquid in collecting pipe is outwelled again.
(7) it is repeated once step 6.
(8) adsorption column with collecting pipe is put into centrifuge, 9000xg sky is from 1min.Collecting pipe is thrown away, and will absorption
Column is placed in sterilized 1.5mL EP pipe.
(9) lid for opening adsorption column, is placed at room temperature for 10min.In order to remove remaining alcohol, otherwise can seriously affect back
Recovery rate and subsequent experimental result.
(10) 15-30 μ L ddH2O is added in adsorbed film center.It is stored at room temperature 5min, 9000xg is centrifuged 1min.1.5mL
The DNA solution that EP bottom of the tube is collected is the DNA recycled, -20 DEG C of preservations.
2.4 DNA recycling segment is connect with PMD-19T carrier
According to PMD19-T carrier operation instructions, by Vector and recycling DNA fragmentation in sterilized PCR reaction tube
T-A clone's connection is carried out, reaction system is as follows:
10h or more is connected overnight for 16 DEG C after mixing gently, convert DH5 α competence.
2.5 connection products convert DH5 α competence
Transformed competence colibacillus is specific steps are as follows: take out appropriate DH5 α competent cell and as freeze thawing on ice.Super
Above-mentioned connection mixed liquor is added into ice-cold competent cell in net workbench, and is gently inhaled with suction pipette head and plays mixing.
It is placed in 20min on ice.Quick and stable is put into 60s in 42 DEG C of water-baths (heat shock keeps cell membrane open, and recombinant plasmid enters cell),
And be put into mixture of ice and water rapidly, it keeps 2min (closing cell membrane).It is added 500 μ L nonreactive LB liquid mediums, 37 DEG C
Constant-temperature shaking culture 1h.4000rpm is centrifuged 5min at room temperature, discards supernatant (retaining a little culture medium, about 50 μ L).Super-clean bench
It is interior, it is gently inhaled with pipette tips and packs resuspended bacterium solution, be applied to ammonia benzyl resistant panel.Agar plate is inverted in 37 DEG C, plate is usually 37
DEG C incubate 14h.
The screening and identification of 2.6 recons
With at least five single colonie (each bacterium colony marks) on 10 μ L trumpet pipette tips picking plates, be placed in containing
In the 1.5mL EP pipe of 800 μ L LB culture mediums (containing corresponding antibiotic), 37 DEG C of shaken cultivation 4h or so to culture medium muddiness, and
Bacterial examination verifying is carried out to the bacterium sample of culture medium muddiness, bacterial examination checking carrier constructs successful bacterium solution and 1mL is taken to serve the raw work sequencing in sea.
Sequence is analyzed using softwares such as BLAST, CLUSTAL, MEGA4.0.
The building of 2.7 expression vectors
Sequencing is obtained into right-on sequence, is carried after extracting plasmid with Kpn I and I double digestion intermediate vector of Xba and expression
Body pCAMBIA13011 (pC13011), then it is connected through with T4 ligase the pC13011 carrier and shiny-leaved yellowhorn of double digestion
Then miRNA169a precursor segment is transformed into Escherichia coli and extracts plasmid enzyme restriction verifying.
2.8 conversion Agrobacteriums
2.8.1 the processing of electric shock cup
(1) ddH is drawn with liquid-transfering gun2O repeated flushing shocks by electricity cup 3-5 times.
(2) it is shocked by electricity cup 3-5 times with 75% ethyl alcohol repeated flushing.
(3) electric shock cup is immersed in 2h in dehydrated alcohol, then outwells ethyl alcohol, is placed in superclean bench and allows residual alcohol
Volatilization.
(4) after alcohol volatilization completely, electric shock cup/lid is covered, room temperature preservation is spare.
2.8.2 electrotransformation
Instrument is used to carry out the conversion of competent cell for Bole company GeneP μ Lser Xcell electroporation apparatus.Main ginseng
Number are as follows: 2.5 μ F of electric pulse, voltage 2.5kV, 200 Ω of resistance.Operating procedure is as follows:
(1) the Agrobacterium GV3101 competent cell placement for taking out preservation is melted on ice.
(2) it takes 1 μ L plasmid to be added in the competent cell of defrosting, mixes gently.
(3) mixed liquor is added in electric shock cup (- 20 DEG C of pre-coolings), is 2.5 μ F, voltage 2.5kV, resistance 200 in electric pulse
It shocks by electricity under the Parameter Conditions of Ω.
(4) electric shock cup is taken out, the YEP fluid nutrient medium of the nonreactive of 800 μ L preheating, after suspension cell, transfer are rapidly added
Into the centrifuge tube of 1.5 mL.
(5) 28 DEG C, 220rpm shake culture 2h or so.
(6) 30-40 μ L bacterium solution is taken, bacterium solution is uniformly coated in containing corresponding antibiotic YEP plate with sterile spreading rod
On, it is inverted into 28 DEG C of incubator cultures 2-3 days.
2.8.3 electrotransformation Agrobacterium and its detection
The correct pC13011-miRNA169a plasmid of digestion verification is transformed into Agrobacterium GV3101, then from Agrobacterium
After middle extraction plasmid, it is transformed into upgrading grain in Escherichia coli, digestion verification.Verifying correctly contains pC13011-miRNA169a
The Agrobacterium of plasmid, -70 DEG C of preservation strains.
2.9 arabidopsis genetic transformations and its homozygous screening of transgenic plant
2.9.1 the plantation of arabidopsis
(1) wildtype Arabidopsis thaliana seed is uniformly sprinkling upon in 1/2MS solid medium.
(2) 4 DEG C of refrigerators are protected from light vernalization treatment 3 days.
After (3) 3 days, culturing room's culture is moved to, condition is 23 DEG C of temperature, light intensity 7000-9000Lx, illumination 16 hours, black
Dark 8 hours.
(4) it after arabidopsis grows 2 cotyledons and 2 true leaves, is transplanted in the basin containing matrix, continues to cultivate.
2.9.2 the genetic transformation of arabidopsis
(1) preparation of Agrobacterium bacterium solution: preparing containing kanamycin and rifampin YEP solid medium, and inverted plate is drawn
Line to activate the Agrobacterium of preservation, cultivate 2 days by 28 DEG C of inversions.It chooses single bacterium and drops down onto YEP fluid nutrient medium of the 1mL containing corresponding antibiotic
In, 28 DEG C are cultivated 2 days, are transferred to and are expanded culture in the identical culture medium of 100mL, until culture solution muddiness becomes orange, are surveyed
Its OD value stops culture when reaching 1.2.
(2) 4000rpm is centrifuged 10min, supernatant is outwelled, with disseminated medium suspension thalline.
(3) flower-dipping method arabidopsis thaliana transformation
A. the arabidopsis to come into bloom is chosen, inflorescence is immersed in the conversion medium containing target gene Agrobacterium, about 10-
20 seconds.
B. the Arabidopsis plant disseminated is lain against in a bulk container, is protected from light culture 24 hours.
C. it second day, places and continues to cultivate under normal condition.
D. arabidopsis seed is harvested, it is dry.
2.9.3 the screening and plantation of arabidopsis transgenic seed
(1) transgenic seed is uniformly sprinkling upon in the solid medium of 1/2MS (concentration of hygromycin is in culture medium
50mg/L).Wildtype Arabidopsis thaliana seed is sprinkling upon without in hygromycin culture medium simultaneously, as control.
(2) 4 DEG C are protected from light vernalization treatment 3 days.
It will be opened sprinkled with the culture dish of wildtype Arabidopsis thaliana seed after (3) 3 days, be placed in culturing room and normally cultivate, and contained
There is the culture dish of transgenic seed to be then protected from light culture.
After (4) 48 hours, the culture dish containing transgenic seed is placed under light and is normally cultivated.The successful plant of transgenosis
It will grow, otherwise not grow.
(5) it after arabidopsis grows 2-4 piece true leaf, is transplanted in the basin containing matrix peat, continues to cultivate.
(6) feelings of blooming of observation control arabidopsis (wild type WT arabidopsis) and shiny-leaved yellowhorn miR169a transgenic arabidopsis
Condition.
3. experimental result
3.1 the Total RNAs extraction of shiny-leaved yellowhorn bud is analyzed
Shiny-leaved yellowhorn bud total serum IgE is extracted using improvement CTAB, and each RNA sample is measured by ultraviolet specrophotometer
The absorbance of 260nm and 280nm, and in the integrality of 1% agarose gel electrophoresis detection RNA.The Reinhoit Zahl of mentioned RNA
Between 2.0-2.2, illustrate pollution of the total serum IgE substantially without carbohydrate, phenol and protein；Electrophoresis result then shows RNA sample
18s and two band of 28s are very clear, and deducibility RNA does not degrade, and meet the requirement of lower step experiment.
The clone of 3.2 shiny-leaved yellowhorn miR169a precursor sequences
According to designed primer sequence, using PCR amplification, product is connected to pMD19-T (simple), bacterial examination PCR
It is detected, and is sent to the raw work sequencing in Shanghai, demonstrate the presence of shiny-leaved yellowhorn miRNA169a precursor.
The functional verification of 3.3 shiny-leaved yellowhorn miR169a
By obtained precursor sequence, the building of expression vector is carried out, miR169a precursor sequence is connected to pC13011 table
It up on carrier, is transformed into DH5 α competent cell, the monoclonal of successful connection is extracted into plasmid, is transformed into soil Agrobacterium
In GV3101, arabidopsis transgenosis is carried out.Resistance screening is carried out to shiny-leaved yellowhorn miR169a transgenic Arabidopsis plants, is screened
After homozygosis, transplanting plantation, seedling observes wild type, 35Spro-miR169a plant phenotype after cultivating 30 days, and discovery is overexpressed
MiR169a plant phenotype is solid compared to WT lines Blooming.
The above is only a preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modifications
Also it should be regarded as protection scope of the present invention.
Sequence table
<110>Forestry Institue, Chinese Inst.of Forestry Sciences
<120>precursor-gene of shiny-leaved yellowhorn miR169a and and its application in flowering of plant in advance
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 171
<212> DNA
<213>artificial sequence
<400> 1
gttaatgagg ttatgttgtg cagccaagga tgacttgccg gcaagctttt atattcattt 60
aacaccaaca ctatatttgt gtgcgcgcgc gtgtttgtat gtgactttag catgatatat 120
gatcattgat cggcaagtcg tctctggcta cattactatc tcattttctc a 171
<210> 2
<211> 21
<212> DNA
<213>artificial sequence
<400> 2
cagccaagga tgacttgccg g 21
<210> 3
<211> 21
<212> DNA
<213>artificial sequence
<400> 3
gttaatgagg ttatgttgtg c 21
<210> 4
<211> 21
<212> DNA
<213>artificial sequence
<400> 4
cattactatc tcattttctc a 21
Claims (10)
1. a kind of precursor-gene of shiny-leaved yellowhorn miR169a, which is characterized in that the sequence of the precursor-gene is as follows:
(a) nucleotide sequence as shown in SEQ ID NO:1, or
(b) at least have 70% with nucleotide sequence shown in SEQ ID NO:1, at least have 75%, at least with 80%, extremely
Have less with 85%, at least with 90%, at least with 95%, at least with 96%, at least with 97%, at least 98% or
At least with 99% homology and the clipped rear nucleotide sequence for generating shiny-leaved yellowhorn miR169a.
2. precursor-gene according to claim 1, which is characterized in that the sequence of the shiny-leaved yellowhorn miR169a is SEQ ID
NO:2.
3. recombinant vector, transgenic cell line or recombinant bacterium containing precursor-gene described in claim 1.
4. recombinant bacterium according to claim 3, which is characterized in that the recombinant bacterium is by precursor base described in claim 1
The recombinant bacterium obtained by insertion expression vector.
5. application of the precursor-gene of shiny-leaved yellowhorn miR169a in flowering of plant in advance, which is characterized in that the shiny-leaved yellowhorn
The sequence of the precursor-gene of miR169a is as follows:
(a) nucleotide sequence as shown in SEQ ID NO:1, or
(b) at least have 70% with nucleotide sequence shown in SEQ ID NO:1, at least have 75%, at least with 80%, extremely
Have less with 85%, at least with 90%, at least with 95%, at least with 96%, at least with 97%, at least 98% or
At least with 99% homology and the clipped rear nucleotide sequence for generating shiny-leaved yellowhorn miR169a.
6. application according to claim 5, which is characterized in that the sequence of the precursor-gene of the shiny-leaved yellowhorn miR169a is
SEQ ID NO:1.
7. application according to claim 5 or 6, which is characterized in that the sequence of the shiny-leaved yellowhorn miR169a is SEQ ID
NO:2.
8. application according to claim 6, which is characterized in that the plant is arabidopsis or tobacco.
9. application according to claim 7, which is characterized in that the plant is arabidopsis.
10. application according to claim 9 characterized by comprising by the precursor comprising the shiny-leaved yellowhorn miR169a
Gene is connected on carrier, by agrobacterium mediation converted to arabidopsis, screening, culture and obtains transgenic line.
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| CN111593058A (en) * | 2020-05-25 | 2020-08-28 | 扬州大学 | Bna-miR169n gene and application thereof in controlling drought resistance of brassica napus |
| CN114716525A (en) * | 2022-04-18 | 2022-07-08 | 大连民族大学 | Xanthoceras sorbifolia dry heat induced transcription factor XsWRKY33 and application thereof |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110373413A (en) * | 2019-06-24 | 2019-10-25 | 浙江农林大学 | The precursor-gene of smoothbark birch miR169a and its application in reduction plant Low nitrogen stress tolerance |
| CN110373413B (en) * | 2019-06-24 | 2023-03-17 | 浙江农林大学 | Precursor gene of Betula luminifera miR169a and application thereof in reducing low-nitrogen stress tolerance of plants |
| CN111593058A (en) * | 2020-05-25 | 2020-08-28 | 扬州大学 | Bna-miR169n gene and application thereof in controlling drought resistance of brassica napus |
| CN114716525A (en) * | 2022-04-18 | 2022-07-08 | 大连民族大学 | Xanthoceras sorbifolia dry heat induced transcription factor XsWRKY33 and application thereof |
| CN114716525B (en) * | 2022-04-18 | 2023-05-26 | 大连民族大学 | Xanthoceras sorbifolia bunge dry heat induction transcription factor XsWRKY33 and application thereof |
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