CN107418954A - Chinese white poplar gene PtomiR390a and its application - Google Patents
Chinese white poplar gene PtomiR390a and its application Download PDFInfo
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Abstract
The invention belongs to field of plant genetic, and in particular to Chinese white poplar genePtomiR390aAnd its application.The invention discloses a kind of Chinese white poplar genePtomiR390a, its nucleotide sequence additionally provides the Chinese white poplar gene as shown in SEQ ID No.1PtomiR390aExpression vector, the host of expression vector, and improve white poplar biomass and improve white poplar salt resistance ability in terms of application.Chinese white poplar gene provided by the inventionPtomiR390aRegulate and control Chinese white poplar biomass in post-transcriptional level, pass through Chinese white poplar genePtomiR390aOverexpression improves the biomass of Chinese white poplar, and improves Chinese white poplar and resist in natural environment by the ability of Saline Alkali Stress.Breeding method provided by the invention, the high-quality Poplar Varieties of high-biomass and high salt resistance ability are cultivated.
Description
Technical field
The invention belongs to field of plant genetic, and in particular to Chinese white poplar gene PtomiR390a and its application.
Background technology
China there are about 1,500,000,000 mu of various salt marsh soils, account for the 26.3% of world's salinized soil area, and Secondary Saline face
Product is still being continuously increased (Yang Jingsong, 2008).The soil salinization turns into the important limitation of China's agriculture and forestry production sustainable development
Factor.Therefore, there is an urgent need to carry out salt tolerant Forest Tree New Varieties seed selection work.Although using traditional breeding technology to a certain extent
The salt tolerance of forest is improved, but the seed selection cycle is long, is difficult to polymerize multiple excellent saline-resisting and alkaline-resisting gene (Luo Zijing in a short time
Deng 2017).And improving forest salt resistance ability using technique for gene engineering has bright prospects.
Willow (Populus spp.) is the most commonly used a kind of fast growing tree of plantation in world wide.At present, China
About 7,000,000 mu of Poplar Plantation area, ranks first in the world (Wang Ying etc., 2007).Under ideal conditions, willow about 4 years is i.e.
It can become a useful person, adaptable strong, reproduction speed soon, easily hybridizes, easily improves the features such as heredity, easy vegetative propagation, is
Important construction timber and paper making raw material, can be used as the raw material of regenerative resource.Since Parsons in 1986 etc. is confirmed
Since willow can carry out genetic transformation, Genetic Engineering of Poplar is developed rapidly.Comospore poplar (Populus
Trichocarpa gene order-checking) completed (Tuskan et al., 2006) in 2006, and this is first is measured
The forest species of genome sequence, therefore willow is counted as the pattern species of xylophyta heredity and molecular breeding research.Hair
White poplar (Populus tomentosa) is the distinctive willow seeds in China, and distributed area (is opened centered on the middle and lower reaches of the Huanghe valley
It is brave etc., 2006).
With the clone progressively goed deep into related resistant gene of salt studied Mechanisms of Salt Resistance, how these gene pairs woodss are utilized
Wood is improved, and turns into focus so as to improve the adaptability of its salinized soil environment.At present, willow salt tolerant transgenic research is main
Concentrate on ion channel and Sodium ransport body, osmotic adjustment gene, protection enzyme gene, transcription factor gene and embryo's hair
Educate late period Abundant protein gene etc. (Luo Zijing etc., 2017).Salt resistance alkali gene mtl-D is transferred to eight by Liu Fenghua etc. (2000)
In village poplar, and screen obtain a collection of transformed plant compared with high-salt tolerance；(2004) such as Sun Zhongxu etc. (2002), Yin Jiandao are to turning
The Ba Li village poplar of mtl-D genes has carried out Field release afforestation experiment, the results showed that the surrival rate of afforestation is bright in moderate saline-alkali soil
It is aobvious to improve.Du etc. (2012) has carried out turning AhDREB1 gene studies to white poplar hybrid, and obtains transgenic line；Liu Xin
(2007) salt tolerance, Wei Bing to turning AhDREB1 gene Chinese white poplars etc. (2009) and Lu etc. (2014) turns to field planting
The security of AhDREB1 gene Chinese white poplars is evaluated.However, these salt tolerant transgenic researches are by other species
The resistant gene of salt of middle identification is transferred in willow, and is unable to reach while is improved forest felling license and the double effectses of salt resistance ability.
MiRNA is a kind of non-coding RNA, and length is about 21 nucleotides, intergenic region is mostly come from, by single-stranded
Precursor forms the hairpin structure of uniqueness in cell.They initially find in Caenorhabditis elegans, find later extensive
It is present in animal and plant cells.MiRNA plays during growth and development of plants is related to stress from outside stimuli responsive
Important regulation and control role, such as developmental regulation and environmental stress react (Chen, 2012).
The content of the invention
The invention provides a kind of Chinese white poplar gene PtomiR390a, and its nucleotide sequence is as shown in SEQ ID No.1.
Present invention also offers the expression vector of the Chinese white poplar gene PtomiR390a.
Specifically, the expression vector is the plant expression vector of overexpression gene.
Specifically, the nucleotide sequence of the plant expression vector is as shown in SEQ ID No.2.
Present invention also offers the host of the expression vector.
Specifically, the host is Agrobacterium.
Present invention also offers the Chinese white poplar gene PtomiR390a to improve white poplar biomass or improve white poplar salt tolerant
Application in terms of ability.
Present invention also offers the expression vector answering in terms of improving white poplar biomass and improving white poplar salt resistance ability
With.
Present invention also offers application of the host in terms of improving white poplar biomass and improving white poplar salt resistance ability.
Present invention also offers one kind application Chinese white poplar gene PtomiR390a to improve Chinese white poplar biomass and salt tolerant
The breeding method of ability, comprises the following steps：
(1) plant expression vector is built：Chinese white poplar gene PtomiR390a is expanded, it is connected with pCXSN carriers, so
Agrobacterium strains GV3101 is converted afterwards；
(2) genetic transformation of Chinese white poplar：Converting material is wild type leaves of Populus Tomentosa, using agriculture bacillus mediated leaf dish
Method；
(3) transfer-gen plant Molecular Detection：Using the genomic DNA of transfer-gen plant as template, resisted by expanding hygromycin
Property marker gene Hyg carry out positive plant screening, filter out the white poplar of high-biomass and salt resistance ability.
Specifically, the nucleotide sequence such as SEQ ID No.3 institutes of amplification Chinese white poplar gene PtomiR390a sense primer
Show, the nucleotide sequence such as nucleotide sequence such as SEQ ID No.4 of anti-sense primer.
Beneficial effects of the present invention：Chinese white poplar gene PtomiR390a provided by the invention is in transcriptional level control Chinese white poplar
Biomass, the biomass of Chinese white poplar is improved by Chinese white poplar gene PtomiR390a overexpressions, and improve Chinese white poplar and support
By the ability of Saline Alkali Stress in imperial natural environment.A kind of application provided by the invention Chinese white poplar gene PtomiR390a is improved
The breeding method of Chinese white poplar biomass and salt resistance ability, the high-quality Poplar Varieties of high-biomass and high salt resistance ability are cultivated.
Brief description of the drawings
Fig. 1：Transfer-gen plant PCR Molecular Detections
With WT strain and transgenic line 35S:PtomiR390a DNA is template, with sequencing primer Hyg-F and
Hyg-R enters performing PCR detection；Wherein M represents Marker DL5000；- it is non-transgenic WT lines.
Fig. 2：Transfer-gen plant expression analysis
WT is wild type Chinese white poplar, PtomiR390a-OXL1, PtomiR390a-OXL2, PtomiR390a-OXL4,
PtomiR390a-OXL7 represents the different strains of PtomiR390a transfer-gen plants respectively.
Fig. 3：Overexpress biomass analysis
A is wild type Chinese white poplar (WT) and overexpression plant (miR390-OE) aerial part phenotype；B is that wild type hair is white
Poplar and overexpression plant under ground portion phenotype；C is wild type Chinese white poplar and overexpression plant plant height；D be wild type Chinese white poplar and
Overexpress plant stem diameter；E is WT lines and overexpression above-ground plant parts dry weight and fresh weight measure；F is wild type
Plant and overexpression plant under ground portion dry weight and fresh weight measure.
Fig. 4：Transfer-gen plant salt resistance is tested
A is 200mM salt stresses processing wild type Chinese white poplar and overexpression plant aerial part phenotype after six weeks；B is 200mM
Salt stress processing wild type Chinese white poplar and overexpression plant under ground portion phenotype after six weeks；C is 200mM salt stresses processing wild type
Counted with Aboveground Biomass of Young after overexpression strain Chinese white poplar six weeks；D is 200mM salt stresses processing wild type and overexpression strain
Be Chinese white poplar after six weeks Underground biomass count.
Embodiment
The willow Total RNAs extraction of embodiment 1 and reverse transcription synthesis cDNA
1st, willow Total RNAs extraction
Using willow spire as material, during RNA is extracted medicine spoon used, pestle, mortar etc. with 95% alcohol calcination to cold
But use afterwards.One spoonful of quartz sand is taken to be used after heating 10min coolings in heater.1.5mL centrifuge tubes, plastic products etc. make
With the AXYGEN products through RNase processing (RNase Free).
Total RNAs extraction is according to magnificent Shun Server-SPIN DNA-free Plant RNA Isolation Kit operating procedures
Carry out：
1) lysate (500 μ L RL+100 μ L 10%PVP+10 μ L 2-ME) is added in 1.5mL centrifuge tubes；
2) willow spire (100mg) and quartz sand are mixed under the conditions of liquid nitrogen frozen and quickly smashed, it is complete in liquid nitrogen
Before full volatilization, powder is transferred completely into the lysate prepared in advance, liquid-transfering gun quickly blows and beats mixing sample；
3) after room temperature places 5min, all move into Filter column, 13400rpm centrifugation 5min, supernatant is all moved into another
In individual centrifuge tube；
4) W3 of supernatant half volume is added, after mixing, adsorption column is all moved into, 12000rpm centrifugation 30s, outwells receipts
Collect liquid in pipe, adsorption column put back in collecting pipe, if cumulative volume more than 750 μ L, upper prop several times；
5) 400 μ L RP are added, after being stored at room temperature 1min, 1min is centrifuged, outwells collection liquid in pipe, adsorption column is put back to
In collecting pipe；
6) 400 μ L W3 liquid are added, after being stored at room temperature 1min, 1min is centrifuged, outwells collection liquid in pipe, adsorption column is put
Reclaim in collector；
7) 40 μ L DNase Ι working solutions are added to adsorbed film center.After being stored at room temperature 15min, 200 μ L RP liquid are added.From
Heart 1min.Collection liquid in pipe is outwelled, adsorption column is put back in collecting pipe；
8) 600 μ L W3 liquid are added, 1min is centrifuged, outwells the liquid in collecting pipe, adsorption column is put back in collecting pipe；
9) 250 μ L W3 liquid are added, stand 1min, 2min is centrifuged, outwells the liquid in collecting pipe, adsorption column is put back into receipts
In collector；
10) adsorption column is moved into a clean 1.5mL centrifuge tube, 50 μ L pure water is added in adsorbed film centre, it is quiet
1min is put, centrifuges 2min.After RNA is slightly mixed, be stored in -70 DEG C it is standby.
2nd, willow total serum IgE reverse transcription synthesis cDNA
Reverse transcription, the reaction system of reverse transcription are carried out with TaKaRa One-step RT PCR Kit kits：
The reaction system of the reverse transcription of table 1
| RNase Free dH2O | 5μL |
| 5×PrimescriptTM Buffer | 2μL |
| Oligo DT Primer | 0.5μL |
| Total RNA | 2μL |
| Primescript RT Enzyme | 2μL |
| Total | 10μL |
The response procedures of reverse transcription：37 DEG C, 15min；85 DEG C, 5s.After terminating reaction, by reaction product in -20 DEG C of storages
It is standby.
Design and the PCR amplification of the PCR primer of embodiment 2 obtain purpose fragment
1st, PCR primer designs
Log in miRBase (http://www.miRBase.com), inquire the precursor sequences of willow PtomiR390a genes
Row.Devise the specific primer of PtomiR390a precursor sequences, the sense primer PtomiR39a0-F of PtomiR390a fragments
Sequence is：5'-AGAATCTGTTAAGCTCAGGAG-3'(SEQ ID No.3), anti-sense primer PtomiR390a-R sequences： 5'-
AACCCATAGAACTCAGGATAG-3'(SEQ ID No.4)；Purpose fragment 119bp.
Primer is by Hua Da gene (Beijing) on behalf of synthesizing.Reaction reagent used in PCR is TaKaRa Products：dNTP
Mixture (each 2.5mM), Taq archaeal dna polymerases (5U/ μ L), attached 10 × PCR Buffer (0.1M Tris-HCl [pH
8.3]), 0.5M KCl) and MgCl2(25mM)。
2nd, PCR expands purpose fragment
Using willow gDNA as template amplification PtomiR390a genetic fragments, the system of PtomiR390a genetic fragments is expanded：
The system of the PCR of table 2 reactions
After adding sample, of short duration centrifugation mixes, and ensures that liquid gathers in ttom of pipe.Foreign gene and endogenous gene are respectively loaded 10
Pipe.It is quickly placed into PCR instrument, following loop parameter is set：94 DEG C of 3min, 1 circulation；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C
1min, 33 circulations.
Amplified production is detected with 1% agarose gel electrophoresis, if purpose band is clear, size is correct, can be according to AxyPrep
The following operating procedure of DNA gel reclaims kits, is reclaimed to purpose band.Cut under uviol lamp containing target DNA
Ago-Gel, exhaust gel surface liquid with paper handkerchief and shred.Calculated for gel weight (records 1.5mL centrifuge tube weights in advance
Amount), the weight is as a gel volume (such as 100mg=100 μ L volumes).Reclaimed by following operation：
1) the Buffer DE-A of 3 gel volumes are added, with 75 DEG C of heating after being well mixed, interruption mixing is (per 2-
3min), until gel piece is completely melt.
2) the Buffer DE-B of 0.5 Buffer DE-A volume are added, are well mixed.When the DNA fragmentation of separation is less than
During 400 bp, the isopropanol of 1 gel volume need to be added (mixture color is changed into yellow after adding Buffer DE-B).
3) mixed liquor in aspiration step 3, is transferred to DNA and prepares in pipe, 12000rpm centrifugation 1min, abandons filtrate.
4) pipe will be prepared and puts back into 2mL centrifuge tubes, added 500 μ LBuffer W1,12000rpm centrifugation 30s, abandon filtrate.
5) pipe will be prepared and puts back into 2mL centrifuge tubes, added 700 μ LBuffer W2,12000rpm centrifugation 30s, abandon filtrate.With same
The method of sample washed once 12000rpm centrifugations 1min with 700 μ LBuffer W2 again.
6) pipe will be prepared and put back into 2mL centrifuge tubes, 12000rpm centrifugations 1min.
7) pipe will be prepared to put with 1.5mL centrifuge tubes, adding 25-30 μ LEluent preparing film center, being stored at room temperature
1min.12000rpm centrifuges 1min eluted dnas.
The PtomiR390a-pcxSN vector constructions of embodiment 3 and conversion Agrobacterium GV3101
After amplified fragments glue reclaim, carrier pcxSN is connected with after XcmI digestions with recovery fragment.Connection product is turned
Change bacillus coli DH 5 alpha competent cell, by PCR amplifying target genes fragment and digestion verification, obtain transformant, and preserve
Strain.
Extract from positive transformant Escherichia coli extraction PtomiR390a-pcxSN plasmids respectively, step according to
Plasmid Mini Kit kit extraction steps, concrete operations are as follows：
1) take culture to the bacterium solution of logarithmic phase in EP pipes, 13400rpm centrifugations 1min collects thalline, abandons supernatant.
2) thalline mixing is resuspended in the μ L of SolutionI 250 for the having added RNase A vortexs for adding precooling.
3) 250 μ L SolutionII actions are added softly to turn upside down 5-6 times, 2min is stood after mixing.
4) 350 μ L SolutionIII are added, turns upside down 5-6 times and occurs to precipitation, 13400rpm centrifuges 10min.
5) supernatant is taken to add adsorption column (about 700 μ L of supernatant), adsorption column is positioned in 2mL collecting pipe, 10000rpm rooms
Temperature centrifugation 1min, abandons liquid.
6) 500 μ L HB Buffer, 10000rpm room temperatures centrifugation 1min is added, abandons liquid.
7) 700 μ L DNA Wash Buffer, 10000rpm room temperatures centrifugation 1min is added, abandons liquid.
8) the 7th step is repeated, 700 μ L DNA Wash Buffer, 10000rpm room temperatures centrifugation 1min is added, abandons liquid.
9) adsorption column is put in collecting pipe, blank pipe 13400rpm centrifugations 2min.
10) adsorption column is put in an other new centrifuge tube, adds 30-50 μ L Elution Buffer, stood
2min, 13400rpm centrifuge 1min.Saved backup in -20 DEG C.
The operation for converting Agrobacterium is as follows：
1) 5 μ L PtomiR390-pcxSN plasmids are taken to be mixed respectively in 200 μ L Agrobacterium GV3101 competent cells
It is even.
2) 30min is placed on ice immediately, is put into liquid nitrogen 2min, 37 DEG C of water-bath 5min rapidly.
3) the empty YEP fluid nutrient mediums of 800 μ L are added, mix rearmounted 28 DEG C, 200rpm cultures 4-6h.
4) 5000rpm centrifuges 8min, abandons 900 μ L of supernatant liquid, and remaining 100 μ L bacterium solutions are mixed, with the coating of sterilization cooling
100 μ L bacterium solutions are uniformly applied on YEP+40mg/L Rif+50mg/L Kan+50mg/L Gen solid mediums by rod, 28 DEG C of bars
Culture 2d is inverted under part.
5) engineering bacteria is named as GV3101-PtomiR390-pcxSN (SEQ ID No.2), adds 15% glycerine to be stored in
- 80 DEG C, tested for follow-up genetic transformation.
The Chinese white poplar genetic transformation of embodiment 4
Converting material is the leaves of Populus Tomentosa of wild type, using Agrobacterium tumefaciens mediated genetic transforming method.
1) activation of Agrobacterium
Agrobacterium GV3101 containing recombinant plasmid is inoculated into YEP solid mediums (Kan+40mg/L containing 50mg/L
Rif on), 28 DEG C of 1~2d of culture；Picking single bacterium colony is inoculated in the YEP fluid nutrient mediums containing 50mg/L Kan, 40mg/L Rif
In, 28 DEG C of shaken cultivations to OD600 are 0.8~1.0；The living bacterial liquids of 200 μ L mono- are taken to go in fresh YEP culture mediums, 28 DEG C are shaken
6~8h of culture is swung, thalline is collected by centrifugation when OD600 reaches 0.6~0.8, with the WPM fluid nutrient mediums 30ml weights that with the addition of AS
1~2h of shaken cultivation in 28 DEG C of shaking tables is put into after outstanding to can be used to convert.
2) conversion of Agrobacterium
The young leaflet tablet on aseptic seedling top is chosen, 0.5 × 0.5cm is cut on superclean bench2Fritter, be put into
10~15min is contaminated in the bacterium solution being resuspended, during which constantly rocks bacterium solution.
3) co-cultivation of Chinese white poplar
The material contaminated carefully is pressed from both sides out to be placed on the flat board containing aseptic filter paper with tweezers and blots bacterium solution, blade is carried on the back
It is laid in downwards on WPM1 solid mediums, the light culture 2d in 25 DEG C of incubators.
4) the selection culture of Chinese white poplar
Transformed explant is moved on on WPM2 Selective agar mediums after 2d, 25 DEG C, illumination be 2000~
Selection culture 3~4 weeks, change a subculture per 10d therebetween in illumination box under the conditions of 10000Lux.
5) culture of sprouting of Chinese white poplar
When occurring white loose callus around blade, move it into and sprout on culture medium WPM3, at 25 DEG C, illumination is
Induction is sprouted in illumination box under the conditions of 2000~10000Lux, about 4~5 weeks time, a subculture is changed per 10d.
6) culture of rootage of Chinese white poplar
When adventitious bud length is to about 2cm, cuts and be transferred in root media WPM4,10d or so is taken root.
7) transplanting of transgenosis Chinese white poplar
When the seedling to take root is grown to about 8cm and more flourishing root system, seedling is taken out, the agar of root is cleaned, is transplanted to
Cultivated in greenhouse.
The transfer-gen plant PCR Molecular Detections of embodiment 5
The resistance regeneration plant of 10~15 plants of transgenosis is chosen respectively, extracts Chinese white poplar genomic DNA.Method is as follows：
1) some sterilized 10ml centrifuge tubes are prepared.One branch is taken, adds 3ml CTAB and 90ul β-mercaptoethanols,
65 DEG C of water-bath preheatings.
2) about 0.3g hairy root is taken, its surface moisture is blotted, is clayed into power in liquid nitrogen, is transferred to the CTAB of above-mentioned preheating
In extract, it is vortexed and mixes.
3) mixed three times in 65 DEG C of water-bath 45min, (soft) vibration in midway interval.
4) isometric Lv Fang ︰ isoamyl alcohol (24 ︰ 1) is added after being cooled to room temperature on ice, gentle inversion keeps flat emulsification 10 after mixing
min.18 DEG C, 10000 revs/min, centrifuge 10min.
5) supernatant (about 2ml) is taken in another clean 10mL centrifuge tubes, adds isometric Lv Fang ︰ isoamyl alcohol (24 ︰ 1),
Gentle inversion keeps flat emulsification 10min after mixing.18 DEG C, 10000 revs/min, centrifuge 10min.
6) supernatant is drawn in another 10mL centrifuge tubes, adds the isopropanol of isometric -20 DEG C of precoolings, and reverse mixing can be shown in
White flock precipitate, amount of short duration can centrifuge less.
7) pipette tips suction out flocculent deposit, and with the rinsing of 500ml 75% (V/V) ethanol twice, 500ml absolute ethyl alcohols rinse again
Once, ethanol is blotted.It is translucent to occurring to be deposited in drying in 37 DEG C of insulating boxs.
8) dissolved and precipitated with 25 μ L sterilized waters, obtain hairy root DNA crude extracts.
9) about 1ul RNase are added into DNA crude extracts, RNA 1h are digested in 37 DEG C.
10) DNA sample, -20 DEG C save backup.
11) template DNA is the genomic DNA of hairy root, is sieved by expanding hpt marker gene Hyg (562bp)
Choosing.The specific primer of Hyg genes is designed, sequence is as follows：
Hyg-F：5′-ATCGGACGATTGCGTCGCATC-3′(SEQ ID No.5)；
Hyg-R：5′-GTGTCACGTTGCAAGACCTG-3′(SEQ ID No.6).
PCR reaction systems are the same as table 2, response procedures：94 DEG C of pre-degeneration 3min, 1 circulation；94 DEG C of denaturation 30s, annealing
30s, 72 DEG C of extension 1min, totally 31 circulations；72 DEG C of extension 10min.1% agarose gel electrophoresis detects amplified production.
The transfer-gen plant expression analysis of embodiment 6
Reverse transcription is square into cDNA after wild type Chinese white poplar is extracted into RNA respectively with the 15 plants of transgenic positive plant obtained
Method designs reference gene PtoU6 primers with embodiment 1：
PtomiR390a stem loop RT：5'-GTCGTATCCAGTGCAGGGTCCGAGGT
ATTCGCACTGGATACGACGGCGCT-3'(SEQ ID No.7)；
PtoU6stem loop RT：5’-GTGCAGGGTCCGAGGTTTTGGACCATTTCT CGAT-3’(SEQ ID
No.7)。
The cDNA of wild type and 1-15 transgenic lines is entered into performing PCR amplification, reaction system is the same as table 2
Response procedures：94 DEG C of pre-degeneration 3min, 1 circulation；94 DEG C of denaturation 30s, anneal 30s, Tm=55 DEG C, 72 DEG C of extensions
1min, the amplification of 25,26,27,28,29,30 periods is carried out successively；72 DEG C of extension 10min.1% agarose gel electrophoresis detects
Amplified production.Cycle-index is selected in linear scope, it can be seen that the period of different strain cDNA concentration differences, and will not
CDNA with concentration difference is diluted to same concentration, and then the cDNA of the wild type of same concentration and overexpression strain is passed through
PCR amplifying target genes, analysis PtomiR390a expression quantity height in different transfer-gen plants, PCR reaction systems are the same as table 2.
It can be seen from Fig. 2, it is higher that screening obtains PtomiR390a-OXL1 strain expression quantity, statistics overexpression biomass.
The transfer-gen plant salt resistance of embodiment 7 is tested
It is 0 (control) to carry out concentration as object, after being cultivated one month in greenhouse using wild type and overexpression plant,
200mmol/L salt stress processing, add concentration respectively by interval 3d for 0 (control), 200mmol/L salt treatment.By interval 7d
Plant plant height and diameter are measured, processing measures plant above ground under ground portion fresh weight after six weeks.
It can be seen from Fig. 3 A, compared to wild type Chinese white poplar plant, overexpress that the blade of plant is bigger, and plant is higher；Ginseng
See that Fig. 3 B understand that the root system of overexpression plant strain is more flourishing.Further, in Fig. 3 C, the plant height of overexpression plant is about
For 1.25 times of wild type Chinese white poplar plant；In Fig. 3 D, the strain stem of overexpression plant is also more sturdy；Fig. 3 E and Fig. 3 F tables
The fresh weight of bright overexpression plant above ground and ground top and the bottom, dry weight are all higher than wild type Chinese white poplar plant.As can be seen here, with it is wild
Type Chinese white poplar plant compares, and the either biomass of overexpression above-ground plant parts or under ground portion is all higher.
It can be seen from Fig. 4 A, handle wild type Chinese white poplar by 200mM salt stresses and overexpress the aerial part table of plant
Type difference is larger, and wild type Chinese white poplar plant major part blade has been wilted, be withered and yellow, and overexpress the only partial blade of plant
Blade tip appearance is withered and yellow, and in Fig. 3 C, the fresh weight for overexpressing above-ground plant parts is about 3 times of wild type Chinese white poplar plant, and dry weight is about
For 2 times, further show, overexpression plant has higher biomass and salt resistance ability；Referring in Fig. 3 B and Fig. 3 D, significantly
It can be seen that the root system for overexpressing plant is more flourishing, biomass is higher.As can be seen here, the situation of 6 weeks is handled in 200mM salinity
Under, significantly wilting does not occur in overexpression plant leaf, and aerial part and root biomass are significantly higher than wild type, and plant is resistance to
Salt ability significantly increases.
Chinese white poplar gene PtomiR390a provided by the invention regulates and controls Chinese white poplar biomass in post-transcriptional level, white by hair
Yankee not only increases the biomass of Chinese white poplar because of PtomiR390a overexpressions, also enhances Chinese white poplar and resists natural environment
In by Saline Alkali Stress ability.Using breeding method provided by the invention, the high-quality of high-biomass and high salt resistance ability has been cultivated
Poplar Varieties.
SEQUENCE LISTING
<110>Southwest University
<120>Chinese white poplar gene PtomiR390a and its application
<130> 8
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 119
<212> DNA
<213>Willow（Populus spp.）
<400> 1
agaatctgtt aagctcagga gggatagcgc catgagcatg acaaagtcta tgtttgagtt 60
aatctcaaca aaatcaatcc agtcatcagt ggcgctatct atcctgagtt ctatgggtt 119
<210> 2
<211> 10232
<212> DNA
<213> Artificial
<220>
<223> PtomiR390a-pcxSN
<400> 2
gaattccgat ctagtaacat agatgacacc gcgcgcgata atttatccta gtttgcgcgc 60
tatattttgt tttctatcgc gtattaaatg tataattgcg ggactctaat cataaaaacc 120
catctcataa ataacgtcat gcattacatg ttaattatta catgcttaac gtaattcaac 180
agaaattata tgataatcat cgcaagaccg gcaacaggat tcaatcttaa gaaactttat 240
tgccaaatgt ttgaacgatc ggggaaattc gctagtggat ccccaatact aacccataga 300
actcaggata gatagcgcca ctgatgactg gattgatttt gttgagatta actcaaacat 360
agactttgtc atgctcatgg cgctatccct cctgagctta acagattcta gtattgggga 420
tccccctcga gtatcgttcg taaatggtga aaattttcag aaaattgctt ttgctttaaa 480
agaaatgatt taaattgctg caatagaagt agaatgcttg attgcttgag attcgtttgt 540
tttgtatatg ttgtgttgag gtcgaggtcc tctccaaatg aaatgaactt ccttatatag 600
aggaagggtc ttgcgaagga tagtgggatt gtgcgtcatc ccttacgtca gtggagatat 660
cacatcaatc cacttgcttt gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc 720
tcgtgggtgg gggtccatct ttgggaccac tgtcggcaga ggcatcttca acgatggcct 780
ttcctttatc gcaatgatgg catttgtagg agccaccttc cttttccact atcttcacaa 840
taaagtgaca gatagctggg caatggaatc cgaggaggtt tccggatatt accctttgtt 900
gaaaagtctc aattgccctt tggtcttctg agactgtatc tttgatattt ttggagtaga 960
caagtgtgtc gtgctccacc atgttatcac atcaatccac ttgctttgaa gacgtggttg 1020
gaacgtcttc tttttccacg atgctcctcg tgggtggggg tccatctttg ggaccactgt 1080
cggcagaggc atcttcaacg atggcctttc ctttatcgca atgatggcat ttgtaggagc 1140
caccttcctt ttccactatc ttcacaataa agtgacagat agctgggcaa tggaatccga 1200
ggaggtttcc ggatattacc ctttgttgaa aagtctcaat tgccctttgg tcttctgaga 1260
ctgtatcttt gatatttttg gagtagacaa gtgtgtcgtg ctccaccatg ttgacctgca 1320
ggcatgcaag cttggcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 1380
ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 1440
aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tgctagagca 1500
gcttgagctt ggatcagatt gtcgtttccc gccttcagtt taaactatca gtgtttgaca 1560
ggatatattg gcgggtaaac ctaagagaaa agagcgttta ttagaataac ggatatttaa 1620
aagggcgtga aaaggtttat ccgttcgtcc atttgtatgt gcatgccaac cacagggttc 1680
ccctcgggat caaagtactt tgatccaacc cctccgctgc tatagtgcag tcggcttctg 1740
acgttcagtg cagccgtctt ctgaaaacga catgtcgcac aagtcctaag ttacgcgaca 1800
ggctgccgcc ctgccctttt cctggcgttt tcttgtcgcg tgttttagtc gcataaagta 1860
gaatacttgc gactagaacc ggagacatta cgccatgaac aagagcgccg ccgctggcct 1920
gctgggctat gcccgcgtca gcaccgacga ccaggacttg accaaccaac gggccgaact 1980
gcacgcggcc ggctgcacca agctgttttc cgagaagatc accggcacca ggcgcgaccg 2040
cccggagctg gccaggatgc ttgaccacct agccctggcg acgttgtgac agtgaccagg 2100
ctagaccgcc tggcccgcag cacccgcgac ctactggaca ttgccgagcg catccaggag 2160
gccggcgcgg gcctgcgtag cctggcagag ccgtgggccg acaccaccac gccggccggc 2220
cgcatggtgt tgaccgtgtt cgccggcatt gccgagttcg agcgttccct aatcatcgac 2280
cgcacccgga gcgggcgcga ggccgccaag gcccgaggcg tgaagtttgg cccccgccct 2340
accctcaccc cggcacagat cgcgcacgcc cgcgagctga tcgaccagga aggccgcacc 2400
gtgaaagagg cggctgcact gcttggcgtg catcgctcga ccctgtaccg cgcacttgag 2460
cgcagcgagg aagtgacgcc caccgaggcc aggcggcgcg gtgccttccg tgaggacgca 2520
ttgaccgagg ccgacgccct ggcggccgcc gagaatgaac gccaagagga acaagcatga 2580
aaccgcacca ggacggccag gacgaaccgt ttttcattac cgaagagatc gaggcggaga 2640
tgatcgcggc cgggtacgtg ttcgagccgc ccgcgcacgt ctcaaccgtg cggctgcatg 2700
aaatcctggc cggtttgtct gatgccaagc tggcggcctg gccggccagc ttggccgctg 2760
aagaaaccga gcgccgccgt ctaaaaaggt gatgtgtatt tgagtaaaac agcttgcgtc 2820
atgcggtcgc tgcgtatatg atgcgatgag taaataaaca aatacgcaag gggaacgcat 2880
gaaggttatc gctgtactta accagaaagg cgggtcaggc aagacgacca tcgcaaccca 2940
tctagcccgc gccctgcaac tcgccggggc cgatgttctg ttagtcgatt ccgatcccca 3000
gggcagtgcc cgcgattggg cggccgtgcg ggaagatcaa ccgctaaccg ttgtcggcat 3060
cgaccgcccg acgattgacc gcgacgtgaa ggccatcggc cggcgcgact tcgtagtgat 3120
cgacggagcg ccccaggcgg cggacttggc tgtgtccgcg atcaaggcag ccgacttcgt 3180
gctgattccg gtgcagccaa gcccttacga catatgggcc accgccgacc tggtggagct 3240
ggttaagcag cgcattgagg tcacggatgg aaggctacaa gcggcctttg tcgtgtcgcg 3300
ggcgatcaaa ggcacgcgca tcggcggtga ggttgccgag gcgctggccg ggtacgagct 3360
gcccattctt gagtcccgta tcacgcagcg cgtgagctac ccaggcactg ccgccgccgg 3420
cacaaccgtt cttgaatcag aacccgaggg cgacgctgcc cgcgaggtcc aggcgctggc 3480
cgctgaaatt aaatcaaaac tcatttgagt taatgaggta aagagaaaat gagcaaaagc 3540
acaaacacgc taagtgccgg ccgtccgagc gcacgcagca gcaaggctgc aacgttggcc 3600
agcctggcag acacgccagc catgaagcgg gtcaactttc agttgccggc ggaggatcac 3660
accaagctga agatgtacgc ggtacgccaa ggcaagacca ttaccgagct gctatctgaa 3720
tacatcgcgc agctaccaga gtaaatgagc aaatgaataa atgagtagat gaattttagc 3780
ggctaaagga ggcggcatgg aaaatcaaga acaaccaggc accgacgccg tggaatgccc 3840
catgtgtgga ggaacgggcg gttggccagg cgtaagcggc tgggttgtct gccggccctg 3900
caatggcact ggaaccccca agcccgagga atcggcgtga cggtcgcaaa ccatccggcc 3960
cggtacaaat cggcgcggcg ctgggtgatg acctggtgga gaagttgaag gccgcgcagg 4020
ccgcccagcg gcaacgcatc gaggcagaag cacgccccgg tgaatcgtgg caagcggccg 4080
ctgatcgaat ccgcaaagaa tcccggcaac cgccggcagc cggtgcgccg tcgattagga 4140
agccgcccaa gggcgacgag caaccagatt ttttcgttcc gatgctctat gacgtgggca 4200
cccgcgatag tcgcagcatc atggacgtgg ccgttttccg tctgtcgaag cgtgaccgac 4260
gagctggcga ggtgatccgc tacgagcttc cagacgggca cgtagaggtt tccgcagggc 4320
cggccggcat ggccagtgtg tgggattacg acctggtact gatggcggtt tcccatctaa 4380
ccgaatccat gaaccgatac cgggaaggga agggagacaa gcccggccgc gtgttccgtc 4440
cacacgttgc ggacgtactc aagttctgcc ggcgagccga tggcggaaag cagaaagacg 4500
acctggtaga aacctgcatt cggttaaaca ccacgcacgt tgccatgcag cgtacgaaga 4560
aggccaagaa cggccgcctg gtgacggtat ccgagggtga agccttgatt agccgctaca 4620
agatcgtaaa gagcgaaacc gggcggccgg agtacatcga gatcgagcta gctgattgga 4680
tgtaccgcga gatcacagaa ggcaagaacc cggacgtgct gacggttcac cccgattact 4740
ttttgatcga tcccggcatc ggccgttttc tctaccgcct ggcacgccgc gccgcaggca 4800
aggcagaagc cagatggttg ttcaagacga tctacgaacg cagtggcagc gccggagagt 4860
tcaagaagtt ctgtttcacc gtgcgcaagc tgatcgggtc aaatgacctg ccggagtacg 4920
atttgaagga ggaggcgggg caggctggcc cgatcctagt catgcgctac cgcaacctga 4980
tcgagggcga agcatccgcc ggttcctaat gtacggagca gatgctaggg caaattgccc 5040
tagcagggga aaaaggtcga aaaggtctct ttcctgtgga tagcacgtac attgggaacc 5100
caaagccgta cattgggaac cggaacccgt acattgggaa cccaaagccg tacattggga 5160
accggtcaca catgtaagtg actgatataa aagagaaaaa aggcgatttt tccgcctaaa 5220
actctttaaa acttattaaa actcttaaaa cccgcctggc ctgtgcataa ctgtctggcc 5280
agcgcacagc cgaagagctg caaaaagcgc ctacccttcg gtcgctgcgc tccctacgcc 5340
ccgccgcttc gcgtcggcct atcgcggccg ctggccgctc aaaaatggct ggcctacggc 5400
caggcaatct accagggcgc ggacaagccg cgccgtcgcc actcgaccgc cggcgcccac 5460
atcaaggcac cctgcctcgc gcgtttcggt gatgacggtg aaaacctctg acacatgcag 5520
ctcccggaga cggtcacagc ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag 5580
ggcgcgtcag cgggtgttgg cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat 5640
agcggagtgt atactggctt aactatgcgg catcagagca gattgtactg agagtgcacc 5700
atatgcggtg tgaaataccg cacagatgcg taaggagaaa ataccgcatc aggcgctctt 5760
ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag 5820
ctcactcaaa ggcggtaata cggttatcca cagaatcagg ggataacgca ggaaagaaca 5880
tgtgagcaaa aggccagcaa aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt 5940
tccataggct ccgcccccct gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 6000
gaaacccgac aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 6060
ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg 6120
tggcgctttc tcatagctca cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca 6180
agctgggctg tgtgcacgaa ccccccgttc agcccgaccg ctgcgcctta tccggtaact 6240
atcgtcttga gtccaacccg gtaagacacg acttatcgcc actggcagca gccactggta 6300
acaggattag cagagcgagg tatgtaggcg gtgctacaga gttcttgaag tggtggccta 6360
actacggcta cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 6420
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt 6480
tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa gatcctttga 6540
tcttttctac ggggtctgac gctcagtgga acgaaaactc acgttaaggg attttggtca 6600
tgcattctag gtactaaaac aattcatcca gtaaaatata atattttatt ttctcccaat 6660
caggcttgat ccccagtaag tcaaaaaata gctcgacata ctgttcttcc ccgatatcct 6720
ccctgatcga ccggacgcag aaggcaatgt cataccactt gtccgccctg ccgcttctcc 6780
caagatcaat aaagccactt actttgccat ctttcacaaa gatgttgctg tctcccaggt 6840
cgccgtggga aaagacaagt tcctcttcgg gcttttccgt ctttaaaaaa tcatacagct 6900
cgcgcggatc tttaaatgga gtgtcttctt cccagttttc gcaatccaca tcggccagat 6960
cgttattcag taagtaatcc aattcggcta agcggctgtc taagctattc gtatagggac 7020
aatccgatat gtcgatggag tgaaagagcc tgatgcactc cgcatacagc tcgataatct 7080
tttcagggct ttgttcatct tcatactctt ccgagcaaag gacgccatcg gcctcactca 7140
tgagcagatt gctccagcca tcatgccgtt caaagtgcag gacctttgga acaggcagct 7200
ttccttccag ccatagcatc atgtcctttt cccgttccac atcataggtg gtccctttat 7260
accggctgtc cgtcattttt aaatataggt tttcattttc tcccaccagc ttatatacct 7320
tagcaggaga cattccttcc gtatctttta cgcagcggta tttttcgatc agttttttca 7380
attccggtga tattctcatt ttagccattt attatttcct tcctcttttc tacagtattt 7440
aaagataccc caagaagcta attataacaa gacgaactcc aattcactgt tccttgcatt 7500
ctaaaacctt aaataccaga aaacagcttt ttcaaagttg ttttcaaagt tggcgtataa 7560
catagtatcg acggagccga ttttgaaacc gcggtgatca caggcagcaa cgctctgtca 7620
tcgttacaat caacatgcta ccctccgcga gatcatccgt gtttcaaacc cggcagctta 7680
gttgccgttc ttccgaatag catcggtaac atgagcaaag tctgccgcct tacaacggct 7740
ctcccgctga cgccgtcccg gactgatggg ctgcctgtat cgagtggtga ttttgtgccg 7800
agctgccggt cggggagctg ttggctggct ggtggcagga tatattgtgg tgtaaacaaa 7860
ttgacgctta gacaacttaa taacacattg cggacgtttt taatgtactg aattaacgcc 7920
gaattaattc gggggatctg gattttagta ctggattttg gttttaggaa ttagaaattt 7980
tattgataga agtattttac aaatacaaat acatactaag ggtttcttat atgctcaaca 8040
catgagcgaa accctatagg aaccctaatt cccttatctg ggaactactc acacattatt 8100
atggagaaac tcgagcttgt cgatcgacag atccggtcgg catctactct atttctttgc 8160
cctcggacga gtgctggggc gtcggtttcc actatcggcg agtacttcta cacagccatc 8220
ggtccagacg gccgcgcttc tgcgggcgat ttgtgtacgc ccgacagtcc cggctccgga 8280
tcggacgatt gcgtcgcatc gaccctgcgc ccaagctgca tcatcgaaat tgccgtcaac 8340
caagctctga tagagttggt caagaccaat gcggagcata tacgcccgga gtcgtggcga 8400
tcctgcaagc tccggatgcc tccgctcgaa gtagcgcgtc tgctgctcca tacaagccaa 8460
ccacggcctc cagaagaaga tgttggcgac ctcgtattgg gaatccccga acatcgcctc 8520
gctccagtca atgaccgctg ttatgcggcc attgtccgtc aggacattgt tggagccgaa 8580
atccgcgtgc acgaggtgcc ggacttcggg gcagtcctcg gcccaaagca tcagctcatc 8640
gagagcctgc gcgacggacg cactgacggt gtcgtccatc acagtttgcc agtgatacac 8700
atggggatca gcaatcgcgc atatgaaatc acgccatgta gtgtattgac cgattccttg 8760
cggtccgaat gggccgaacc cgctcgtctg gctaagatcg gccgcagcga tcgcatccat 8820
agcctccgcg accggttgta gaacagcggg cagttcggtt tcaggcaggt cttgcaacgt 8880
gacaccctgt gcacggcggg agatgcaata ggtcaggctc tcgctaaact ccccaatgtc 8940
aagcacttcc ggaatcggga gcgcggccga tgcaaagtgc cgataaacat aacgatcttt 9000
gtagaaacca tcggcgcagc tatttacccg caggacatat ccacgccctc ctacatcgaa 9060
gctgaaagca cgagattctt cgccctccga gagctgcatc aggtcggaga cgctgtcgaa 9120
cttttcgatc agaaacttct cgacagacgt cgcggtgagt tcaggctttt tcatatctca 9180
ttgccccccg gatctgcgaa agctcgagag agatagattt gtagagagag actggtgatt 9240
tcagcgtgtc ctctccaaat gaaatgaact tccttatata gaggaaggtc ttgcgaagga 9300
tagtgggatt gtgcgtcatc ccttacgtca gtggagatat cacatcaatc cacttgcttt 9360
gaagacgtgg ttggaacgtc ttctttttcc acgatgctcc tcgtgggtgg gggtccatct 9420
ttgggaccac tgtcggcaga ggcatcttga acgatagcct ttcctttatc gcaatgatgg 9480
catttgtagg tgccaccttc cttttctact gtccttttga tgaagtgaca gatagctggg 9540
caatggaatc cgaggaggtt tcccgatatt accctttgtt gaaaagtctc aatagccctt 9600
tggtcttctg agactgtatc tttgatattc ttggagtaga cgagagtgtc gtgctccacc 9660
atgttatcac atcaatccac ttgctttgaa gacgtggttg gaacgtcttc tttttccacg 9720
atgctcctcg tgggtggggg tccatctttg ggaccactgt cggcagaggc atcttgaacg 9780
atagcctttc ctttatcgca atgatggcat ttgtaggtgc caccttcctt ttctactgtc 9840
cttttgatga agtgacagat agctgggcaa tggaatccga ggaggtttcc cgatattacc 9900
ctttgttgaa aagtctcaat agccctttgg tcttctgaga ctgtatcttt gatattcttg 9960
gagtagacga gagtgtcgtg ctccaccatg ttggcaagct gctctagcca atacgcaaac 10020
cgcctctccc cgcgcgttgg ccgattcatt aatgcagctg gcacgacagg tttcccgact 10080
ggaaagcggg cagtgagcgc aacgcaatta atgtgagtta gctcactcat taggcacccc 10140
aggctttaca ctttatgctt ccggctcgta tgttgtgtgg aattgtgagc ggataacaat 10200
ttcacacagg aaacagctat gaccatgatt ac 10232
<210> 3
<211> 21
<212> DNA
<213> Artificial
<220>
<223> PtomiR390a-F
<400> 3
agaatctgtt aagctcagga g 21
<210> 4
<211> 21
<212> DNA
<213> Artificial
<220>
<223> PtomiR390a-R
<400> 4
aacccataga actcaggata g 21
<210> 5
<211> 21
<212> DNA
<213> Artificial
<220>
<223> Hyg-F
<400> 5
atcggacgat tgcgtcgcat c 21
<210> 6
<211> 20
<212> DNA
<213> Artificial
<220>
<223> Hyg-R
<400> 6
gtgtcacgtt gcaagacctg 20
<210> 7
<211> 50
<212> DNA
<213> Artificial
<220>
<223> PtomiR390a stem loop RT
<400> 7
gtcgtatcca gtgcagggtc cgaggtattc gcactggata cgacggcgct 50
<210> 8
<211> 34
<212> DNA
<213> Artificial
<220>
<223> PtoU6 stem loop RT
<400> 8
gtgcagggtc cgaggttttg gaccatttct cgat 34
Claims (10)
1. Chinese white poplar genePtomiR390a, its nucleotide sequence is as shown in SEQ ID No.1.
2. Chinese white poplar gene according to claim 1PtomiR390aExpression vector.
3. expression vector according to claim 2, it is characterised in that：The expression vector is the plant of overexpression gene
Expression vector.
4. expression vector according to claim 3, it is characterised in that：The nucleotide sequence of the plant expression vector is such as
Shown in SEQ ID No.2.
5. according to the host of any one of the claim 2-4 expression vectors, it is preferred that the host is Agrobacterium.
6. Chinese white poplar gene according to claim 1PtomiR390aImprove white poplar biomass and improve white poplar salt resistance ability
The application of aspect.
7. according to any one of the claim 2-4 expression vectors in terms of improving white poplar biomass and improving white poplar salt resistance ability
Application.
8. according to any one of the claim 5-6 hosts answering in terms of improving white poplar biomass and improving white poplar salt resistance ability
With.
9. one kind applies the Chinese white poplar genePtomiR390aImprove the breeding method of Chinese white poplar biomass and salt resistance ability, bag
Include following steps：
（1）Build plant expression vector：Expand Chinese white poplar genePtomiR390a, it is connected with pCXSN carriers, Ran Houzhuan
Change agrobacterium strains GV3101；
（2）The genetic transformation of Chinese white poplar：Converting material is wild type leaves of Populus Tomentosa, using agriculture bacillus mediated leaf disk method；
（3）Transfer-gen plant Molecular Detection：Using the genomic DNA of transfer-gen plant as template, by expanding hygromycin resistance mark
Remember geneHygPositive plant screening is carried out, filters out the white poplar of high-biomass and salt resistance ability.
10. breeding method according to claim 10, it is characterised in that amplification Chinese white poplar genePtomiR390aUpstream draw
The nucleotide sequence of thing is as shown in SEQ ID No.3, the nucleotide sequence such as nucleotide sequence such as SEQ ID of anti-sense primer
No.4。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
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| CN111304198B (en) * | 2019-12-13 | 2020-10-09 | 南京林业大学 | Application of cymbidium goeringii miR390b in controlling plant vegetative organ development |
| CN116254290A (en) * | 2022-08-03 | 2023-06-13 | 西南大学 | Application of PtoPLT5a gene in improving biomass and fiber cell length of populus tomentosa |
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