CN109055371A - The precursor-gene of smoothbark birch miR169c and its application in flowering of plant in advance - Google Patents
The precursor-gene of smoothbark birch miR169c and its application in flowering of plant in advance Download PDFInfo
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Abstract
The invention discloses the precursor-genes of smoothbark birch miR169c 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 smoothbark birch miR169c of generation afterwards.The present invention provides application of the precursor-gene of smoothbark birch miR169c in flowering of plant in advance.Using the precursor-gene of smoothbark birch miR169c, it is expected to artificially shift to an earlier date flowering of plant.
Description
Technical field
The present invention relates to the precursor-gene of smoothbark birch miR169c a kind of and its applications 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 mistake by inhibiting NF-YA genetic transcription
Expression promotes arabidopsis Blooming.However, the function of smoothbark birch miR169c is on the knees of the gods.
Summary of the invention
The object of the present invention is to provide the precursor-gene of smoothbark birch miR169c 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 smoothbark birch miR169c.
In one embodiment, the sequence of the smoothbark birch miR169c is SEQ ID NO:2.
The present invention also provides the recombinant vectors, transgenic cell line or recombinant bacterium of having given above-mentioned precursor-gene.
In one embodiment, the recombinant bacterium is the recombination that precursor-gene described above is inserted into that expression vector obtains
Bacterium.
The present invention also provides given application of the above-mentioned precursor-gene in flowering of plant in advance.
In one embodiment, the plant is arabidopsis or tobacco.
In one embodiment, the plant is arabidopsis.
In one embodiment, above-mentioned application includes being connected to the precursor-gene comprising the smoothbark birch miR169c
On carrier, by agrobacterium mediation converted to arabidopsis, screening, culture and transgenic line is obtained.
The present invention provides application of the precursor-gene of smoothbark birch miR169c in flowering of plant in advance.Using smoothbark birch
The precursor-gene of miR169c is expected to artificially shift to an earlier date flowering of plant.
Detailed description of the invention
The drawings described herein are used to provide a further understanding of the present application, constitutes part of this application, this Shen
Illustrative embodiments and their description please are not constituted an undue limitation on the present application for explaining the application.In the accompanying drawings:
Fig. 1 is the total serum IgE of smoothbark birch blade of the invention；
Fig. 2 is that the expression vector bacterium solution PCR of smoothbark birch miR169c precursor is detected；
Fig. 3 is the resistance screening of smoothbark birch miR169c transgenic Arabidopsis plants seed；
Fig. 4 is the arabidopsis PCR detection for being overexpressed miR169c；
Fig. 5 is that (Col-0 is open country for the flowering phenotype of the wild type observed and the arabidopsis of smoothbark birch miR169c transgenosis
Raw type, OEmiR169c is transgenic plant).
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, with reference to the accompanying drawing and specific implementation
Invention is further described in detail for example.It should be appreciated that described herein, specific examples are only used to explain the present invention, and
It is not used in the restriction present invention.
1. material
1.1 experimental material
Experimental material is derived from the tissue-cultured seedling of Zhejiang Province Linan Zhejiang A & F University Development of intelligent laboratory, kind smoothbark birch G49-3,
Sample adopt it is lower after Liquid nitrogen storage immediately, be placed on -80 DEG C of refrigerators save it is stand-by.Arabidopsis wild type seeds are raw using Colombia
State type (Columbia-0).
1.2 experiment reagents and instrument
Archaeal dna polymerase, various restriction enzymes, T4 ligase, Marker and TRIzol reagent are purchased from precious biological work
Journey (Dalian) Co., Ltd；Plasmid extraction kit and DNA plastic recovery kit are limited purchased from the raw work bioengineering share in Shanghai
Company.PCR instrument is U.S. PE9700PCR instrument, and superclean bench really purifies Science and Technology Ltd. purchased from Suzhou.
The synthesis of 1.3 primers and sequencing
Primer synthesis and sequencing are completed by Sangon Biotech (Shanghai) Co., Ltd..
2. method
The extraction of 2.1 smoothbark birch total serum IgEs
The RNA (Total RNA) of smoothbark birch blade is extracted using improvement CTAB+Trizol 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 blade 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 12000rpm4 DEG C of centrifugation 10min；
(6) it is primary to repeat step 4, until middle layer disappears；
(7) 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 complete molten in precipitating；
(8) 1mLTRIzol reagent is added in solution, is stored at room temperature 5min.It adds 200 μ L chloroforms to shake up, be stored at room temperature
2-3min；
(9) 4 DEG C of 12000rpm are centrifuged 15min；
(10) supernatant is drawn, isometric isopropanol is added, is stored at room temperature 10min；
(11) 4 DEG C of 12000rpm are centrifuged 10min, discard supernatant, and the visible RNA of naked eyes is sunken to tube bottom；
(12) the 75% ethanol washing precipitating of 1mL pre-cooling is added, mild to vibrate, 8000rpm4 DEG C of centrifugation 5min is discarded
Clearly；
(13) step 11 is repeated, and precipitating is dried in vacuo 7-10min；Appropriate DEPC water dissolution precipitating, -80 DEG C of guarantors are added
It deposits spare.
2.2 gene cloning
After the RNA (Total RNA) of smoothbark birch is diluted with appropriate DEPC ddH2O, referring to PrimeScriptTM RT
The specification of Reagent Kit (Perfect Real Time) (TaKaRa) carries out the synthesis of the first chain of reverse transcription cDNA.It prepares
Following gene cloning PCR system (20ul):
Response procedures are as follows: 94 DEG C, 3min；It 94 DEG C, 30sec, 58 DEG C, 30sec, 72 DEG C, 1.5min, recycles 37 times；72℃,
8min。
Gene cloning the primer (table 1).
The recycling of 2.3 target fragments
Utilize AxyPrepTMDNA gel QIAquick Gel Extraction Kit (AXYGEN) carries out target fragment recycling.
(1) Ago-Gel containing gene target fragment is cut in the UV lamp, weighs gel weight, and the weight is as one
A gel volume (such as 100mg=100ul).
(2) Buffer DE-A solution is added, about 3 gel volumes are put into 75 DEG C of water-baths and heat 6-8min, during which every
It is rocked 1 time every 2-3min, until gel melts completely.
(3) Buffer DE-B solution is added, about 1/2 Buffer DE-A volume mixes；When target fragment length is less than
When 400bp, appropriate isopropanol, about 1 gel volume are added.
(4) mixed liquor of previous step is drawn onto DNA preparation pipe (being placed in 2ml centrifuge tube), 12000rpm1min goes to filter
Liquid.
(5) plus 500ul Buffer W1 is in pipe of purchasing, 12 000rpm 1min, abandons filtrate.
(6) plus 700ul Buffer W2 is in pipe of purchasing, 12 000rpm 1min, abandons filtrate, is repeated 1 times.
(7) pipe that will purchase is placed in 2ml centrifuge tube, 12 000rpm 1min, abandons filtrate.
(8) pipe of purchasing is put into clean 1.5ml centrifuge tube, pipe center adds 25-30ul to be pre-heated to 65 DEG C to purchasing
DdH2O is stored at room temperature 1-3min, 12 000rpm 1min, eluted dna.
(9) 2-3ul DNA solution is taken to be detected for agarose gel electrophoresis, surplus solution places -20 DEG C of preservations.
The connection of 2.4 target fragments
Following solutions are mixed, are mixed gently, of short duration centrifugation.PCR:25 DEG C of 5-30min is (according to target gene size
It is fixed).
The conversion of 2.5 target fragments
(1) it by 5ul connection product, is added in 50ul competence TransT1, ice bath 25-30min.
(2) 42 DEG C of water-bath heat shock 40sec.It is placed in 2min on ice.
(3) 300-500ul is added without hormone LB liquid medium, 37 DEG C of culture 30-60min of 200rpm.
(4) 4000rpm 2min removes part supernatant, stays 50-100 μ l bacterium solution, and 10 μ l isopropylthiogalactosides are added
(IPTG, 100mg/ml) and the 8 chloro- 3- indoles-α-D- galactosides (X-gal, 100mg/ml) of the bromo- 4- of μ l 5- mix.
(5) remaining solution is applied to kanamycins (Kan, 50mg L-1) in solid medium, 37 DEG C of inversions trainings
It supports overnight.
2.6 target fragment PCR verifying
(1) in picking 1.3.2 step 5 white single colonie, be placed in the LB liquid medium 500ul of same concentrations antibiotic
In, 37 DEG C of culture 3-6h take 1ul, are used for bacterium solution PCR.
(2) bacterium solution PCR detection is carried out with gene cloning primer, reaction system (10ul) is as follows:
(3) PCR program are as follows: 94 DEG C of 3min；94 DEG C of 30sec, 57 DEG C of 30sec, 72 DEG C of x min are (according to Insert Fragment
Length is determined), it recycles for 30 times；72℃ 10min.
(4) agarose gel electrophoresis detects PCR product, and selection positive colony, which is sent to raw work bioengineering (Shanghai) share, to be had
The sequencing of limit company.
The BlmiR169 precursor premiR 169c obtained using above-mentioned clone, constructs the overexpression vector of miR169.
The building of 2.7 entry vectors
According to pCRTM8/GW/Kit (Invitrogen) kit specification building introduction carries
Body.
(1) following PCR system (20ul) is prepared:
(2) PCR program are as follows: 94 DEG C of 5min；94 DEG C of 30sec, 58 DEG C of 30sec, 72 DEG C of 1min are recycled 35 times；72℃
5min。
(4) guarantee product after single band, to take preparation of the 1ul PCR product for following component, remaining PCR product is protected
It is stored in -20 DEG C of refrigerators.
(5) above-mentioned mixed liquor is mixed, and of short duration centrifugation.Then it is according to condition reacted in PCR instrument: 25 DEG C of 5-20min.
(6) reaction solution in 2ul step 5 is taken, is added in 50ul Trans1T1 competence, soft to mix, ice bath 25-
30min。
(7) it is placed in 42 DEG C of water-baths heat shock 40 seconds, adds 250ul fluid nutrient medium, 37 DEG C of incubation 45- of 200rpm
60min。
(8) 4000rpm is centrifuged 3min and removes part supernatant, and remaining bacterium solution is applied to spectinomycin (Spec, 50mg L-1)
LB solid medium in, overnight incubation.
(9) bacterium solution PCR detection is carried out with gene specific primer, and confirmation is sequenced.
2.8 expression buildings
That genetic transformation expression carrier is selected is pGWB8 (35S pro, C-6xHis), is constructed MiR169 (BlmiR169c)
Expression vector.
(1) following mixed solution, 25 DEG C of 1h, 72 DEG C of 10min are prepared；
(2) take 2ul for connect convert, then choose clone, bacterium solution PCR is detected；
(3) PCR system:
(4) PCR program: 94 DEG C of 5min；94 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of x min are (according to target gene piece
Segment length) circulation 35 times；72℃ 5min；
(5) agarose gel electrophoresis detects PCR product, and positive colony is chosen after conversion to Escherichia coli and is sent to raw work biology
The sequencing of engineering (Shanghai) limited liability company；
(6) the correct bacterial strain plasmid of sequencing result is extracted, for use.
2.9 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) YEP solid and fluid nutrient medium (table) are configured
(2) add rifampin and kanamycins antibiotic (50mg.L when YEP solid medium is micro- cool-1Rif and 50mg.L- 1), Kan until inverted plate on superclean bench.
(3) the Agrobacterium GV3101 competent cell placement taken out in -80 DEG C of Storage in refrigerator is melted on ice；
(4) it takes 1 μ L recombinant plasmid to be added in the competent cell of defrosting, mixes gently；
(5) ddH is used2The ethyl alcohol of O and 75% cleans electric shock cup 3-5 times repeatedly, and superclean bench dries；
(6) the Agrobacterium GV3101 competent cell placement taken out in -80 DEG C of Storage in refrigerator is melted on ice；
(7) it takes 1 μ L recombinant plasmid to be added in the competent cell of defrosting, mixes gently；
(8) 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；
(9) 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 1.5mL；
(10) 28 DEG C, 220rpm shake culture 2h or so；
(11) 40 μ L bacterium solutions are taken, are uniformly coated on the YEP plate containing rifampin and kalamycin resistance, inversion is put
Enter 28 DEG C of incubator culture 2-3d.
2.10 electrotransformation Agrobacteriums and its detection
The Escherichia coli of the 35Spro-miR169 containing expression vector extract plasmid and are transformed into Agrobacterium GV3101, then from agriculture
After extracting plasmid in bacillus, detected using gene primer.Agrobacterium of the verifying correctly containing 35Spro-miR169 plasmid, -70
DEG C save strain.
The operating procedure of 2.11 inflorescence dip method arabidopsis thaliana transformations
(1) (COL-0) program request of wildtype Arabidopsis thaliana seed moves to when length to four cotyledons in 1/2MS culture medium
In the basin of sterile soil cave, cultivated in culturing room.Condition of culture are as follows: daytime 24 DEG C/15h, 24 DEG C/9h of night.It subtracts within 3 weeks or so quasi-
Southern mustard stem inhibits apical dominance to promote reproductive growth.Inflorescence is used after arabidopsis extracts a plurality of side shoot out and enters full-bloom stage
Dip method infects arabidopsis.Soil is impregnated with before infecting.
(2) arabidopsis infiltration medium (1/2MS fluid nutrient medium, PH5.7) is configured.It will be containing the agriculture for having purpose expression vector
Bacillus draws plate activation (the YEP culture medium for having antibiotic), and picking monoclonal is inoculated in the YEP Liquid Culture of 200mL added with antibiotic
Base (50mg.L-1Rif and 50mg.L-1Kan), 28 DEG C of constant-temperature tables, 200rpm cultivate 2d, until final concentration OD600=0.8-1.0.
(3) it collecting bacterial strain: bacterium solution is poured into 50mL sterile centrifugation tube, 4000-6000rpm is centrifuged ten minutes, supernatant is abandoned,
Thallus is resuspended with isometric infiltration medium, and 0.02% surfactant silwet is added.
(4) by the arabidopsis floral of full-bloom stage, be soaked in gently dry plant leaf in re-suspension liquid after 40 seconds leave liquid
Body.
(5) plant after infecting puts on polybag, and dark culture 12-24h supports 7-10d later, carries out Secondary Infection.
(6) plant after infecting, normal to cultivate, 3 days reduction irrigation amounts before seed is mature are collected T0 by single plant and are filled for seed
Enter in 1.5mL centrifuge tube, deposits in 4 DEG C of refrigerators.
2.12 arabidopsis transgenic positive strains screening
(1) experiment reagent: 75% ethyl alcohol, dehydrated alcohol, sterile water, the 1/2MS solid medium containing antibiotic
(Hyg concentration is 30mgL-1)
(2) suitable transgenic arabidopsis seed to be screened is taken, is added in the centrifuge tube of 1.5Ml, 75% ethyl alcohol is added,
Sufficiently oscillation 10min.After going upper solution, dehydrated alcohol is added and vibrates 1min, repetitive operation is three times.In the filter of sterilizing
Paper is dried up in superclean bench.
(3) it by the arabidopsis seed of drying, is uniformly seeded in solid medium, seals culture dish, 4 DEG C of vernalization cultures
1d, after be placed in long-day incubator (sunshine 16h/ night 8h).
(4) arabidopsis germination in culture dish is observed, until selection root system is good when seedling has 4-5 piece cotyledon, blade edge
The seedling of color, transplanting are grown into matrix.
(5) when arabidopsis has certain biomass, leaf DNA is extracted, carries out positive detection.
(6) growth of observation control arabidopsis (wild type Col-0 arabidopsis) and smoothbark birch miR169c transgenic arabidopsis
Situation.
3. experimental result
The blade Total RNAs extraction of 3.1 smoothbark birches is analyzed
The total serum IgE (see Fig. 1) of smoothbark birch blade is extracted using improvement CTAB+TRizol method, and passes through uv-spectrophotometric
Meter measures the absorbance of each RNA sample 260nm and 280nm, and the concentration and purity of RNA is calculated with this, and in 1% agarose
The integrality of detected through gel electrophoresis RNA.The Reinhoit Zahl of mentioned RNA illustrates the basic sugar-free of total serum IgE between 2.0-2.2
The pollution of class, phenol and protein；Electrophoresis result then shows that two band of 18s and 28s of RNA sample is very clear, and deducibility RNA does not have
There is degradation, meets the requirement of lower step experiment.
The clone of 3.2 smoothbark birch miR169c precursor sequences
According to designed primer sequence, using PCR amplification, bacterial examination PCR is detected (Fig. 2), and is sent to raw work biology
The sequencing of engineering (Shanghai) limited liability company, demonstrates the presence of smoothbark birch miR169c precursor.
The functional verification of 3.3 smoothbark birch miR169c
By obtained precursor sequence, the building of expression vector is carried out, smoothbark birch miR169c precursor sequence is connected to
It on pC13011 expression vector, is transformed into DH5 α competent cell, the monoclonal of successful connection is extracted into plasmid, is transformed into soil
In earth Agrobacterium GV3101, arabidopsis transgenosis is carried out.Resistance screening is carried out to smoothbark birch miR169c Arabidopsis plant seed
The transgenic arabidopsis body filtered out is taken DNA by (such as Fig. 3), and carrying out PCR detection, (such as Fig. 4, PCR amplification obtain smoothbark birch
MiR169c precursor-gene).Transplanting plantation, seedling observe wild type, 35Spro-miR169c plant phenotype, hair after cultivating 20 days
It is solid (Fig. 5) compared to WT lines Blooming to be now overexpressed miR169c.
The above description is merely a specific embodiment, but scope of protection of the present invention is not limited thereto, any
Those familiar with the art in the technical scope disclosed by the present invention, can easily think of the change or the replacement, and should all contain
Lid is within protection scope of the present invention.Therefore, protection scope of the present invention should be subject to the protection scope in claims.
Sequence table
<110>Zhejiang A & F University
<120>precursor-gene of smoothbark birch miR169c and its application in flowering of plant in advance
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 174
<212> DNA
<213>artificial sequence
<400> 1
ctcgcatgta cggcgtagaa agtattatgc agccaaggat gacttgccga ctcttgctag 60
ctaggtagtt tgtttcctct ttataattca agtagtacta ctgatccaaa aaccgatcga 120
tcaaccaagt cggcaagttg tcgttggcta catgtttctt tcttctccgc atgc 174
<210> 2
<211> 21
<212> DNA
<213>artificial sequence
<400> 2
agccaaggat gacttgccga c 21
<210> 3
<211> 21
<212> DNA
<213>artificial sequence
<400> 3
ctcgcatgta cggcgtagaa a 21
<210> 4
<211> 21
<212> DNA
<213>artificial sequence
<400> 4
gcatgcggag aagaaagaaa c 21
Claims (8)
1. a kind of precursor-gene of smoothbark birch miR169c, 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 smoothbark birch miR169c.
2. precursor-gene according to claim 1, which is characterized in that the sequence of the smoothbark birch miR169c 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 described in any one of claim 1-4 in flowering of plant in advance.
6. application according to claim 5, which is characterized in that the plant is arabidopsis or tobacco.
7. application according to claim 6, which is characterized in that the plant is arabidopsis.
8. application according to claim 7 characterized by comprising by the precursor base comprising the smoothbark birch miR169c
Because being connected on carrier, by agrobacterium mediation converted to arabidopsis, screening, culture and transgenic line is obtained.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| 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 |
| CN114214325A (en) * | 2021-11-26 | 2022-03-22 | 浙江农林大学 | Betula luminifera miR156a precursor gene and application thereof in promoting plant branch formation |
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| CN105420273A (en) * | 2016-01-04 | 2016-03-23 | 东北农业大学 | Method for cultivating transgenic plants with blooming ahead of time |
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| CN105420273A (en) * | 2016-01-04 | 2016-03-23 | 东北农业大学 | Method for cultivating transgenic plants with blooming ahead of time |
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Cited By (4)
| 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 |
| CN114214325A (en) * | 2021-11-26 | 2022-03-22 | 浙江农林大学 | Betula luminifera miR156a precursor gene and application thereof in promoting plant branch formation |
| CN114214325B (en) * | 2021-11-26 | 2023-11-17 | 浙江农林大学 | Betula alnoides miR156a precursor gene and application thereof in promotion of plant branching formation |
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