CN108753795A - A kind of gene NtNHX1-3 improving tobacco leaf potassium content and its cloning process and application - Google Patents
A kind of gene NtNHX1-3 improving tobacco leaf potassium content and its cloning process and application Download PDFInfo
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Abstract
The invention discloses a kind of gene NtNHX1-3 improving tobacco leaf potassium content and its cloning process and applications, improve the gene NtNHX1-3 nucleotide sequences such as SEQ ID of tobacco leaf potassium content：Shown in No.1, the amino acid sequence such as SEQ ID of coding：Shown in No.2.The invention also discloses the cloning process for the gene NtNHX1-3 for improving tobacco leaf potassium content, specific steps include：A, NtNHX1-3 gene orders are determined；B, tobacco RNA is extracted, reverse transcription obtains the first chain cDNA；C, synthesis specific primer is designed according to NtNHX1-3 gene orders, using cDNA as template, carries out PCR amplification；D, recycling and purified pcr product；Construct the over-express vector of the gene containing NtNHX1-3.Over-express vector is overexpressed by Agrobacterium-medialed transformation in tobacco, prepare transgenosis plant.The transfer-gen plant potassium content of acquisition is significantly improved than control.Illustrate that tobacco NtNHX1-3 genes have great application prospect in terms of the tobacco for cultivating high potassium content.
Description
Technical field
The invention belongs to biotechnologies, and in particular to it is a kind of improve tobacco leaf potassium content gene NtNHX1-3 and
Its cloning process and application.
Background technology
Plant mainly passes through the Na on cell membrane or tonoplast+/H+Antiporter protein maintains Na in cytoplasm+And K+
Concentration.ArabidopsisAtNHX1The clone of gene and functional verification show that NHX1 plays Na in plant+/H+Antiport function
（Apse et al., 1999, Science, 285:1256-1258）.Due to NHX albumen in statocyte Na+The work of concentration
With, can be with after being overexpressed AtNHX1 genes in terms of being concentrated mainly on plant salt tolerance stress to the research of its biological function at present
Significantly improve arabidopsis（Apse et al., 1999, Science, 285:1256-1258））, rape（Zhang et al.,
2001, Proc. Natl. Acad. Sci. USA, 98:12832-12836）And tomato（Zhang and Blumwald.,
2001, Nat. Biotechnol. 19:765-768）Salt tolerance.It is being overexpressedAtNHX1Transgene tomato in, AtNHX1
Also it is demonstrated by K+/H+Transport body function（Zhang and Blumwald., 2001, Nat. Biotechnol. 19:765-
768）.AtNHX1 and AtNHX2 Gene Doubles mutating strain series cannot be by K+It is pumped into vacuole, while making cytosol K+Concentration increases（Barragá
N et al., 2012, Plant Cell, 24 (3):1127-42）.Show two transporters of AtNHX1 and AtNHX2 in K+From
Cytosol, which is pumped into vacuole, plays key effect.
Potassium is considered as the Quality Element of tobacco, and potassium content is one of the important indicator for evaluating quality of tobacco quality.Tobacco leaf
Potassium content improves the institutional framework that can improve tobacco leaf, keeps tobacco leaf structure fine and smooth, and can also improve tobacco leaf appearance luster, makes cigarette
Leaf is in deep crocus, fragrance foot, jealous good, high resilience and toughness, fillibility enhancing.In addition potassium can also enhance tobacco leaf sugar
The dynamic accumulation of class, pigment, aromatic substance.
Invention content
The first object of the present invention is to provide a kind of gene NtNHX1-3 improving tobacco leaf potassium content；Second purpose
It is to provide the cloning process of the gene NtNHX1-3 of the raising tobacco leaf potassium content；Third is designed to provide described
Raising tobacco leaf potassium content gene NtNHX1-3 application.
The first object of the present invention is achieved in that the gene NtNHX1-3's of the raising tobacco leaf potassium content
Nucleotide sequence such as sequence table SEQ ID NO：Shown in 1.
The second object of the present invention, which is achieved in that, to be included the following steps：
A, NtNHX1-3 gene orders are determined；
Ncbi database, which is searched for, according to the protein sequence of rice NHX1 genes obtains homologous gene NtNHX1-3 sequences in tobacco, profit
With this sequence design gene cloning primer：
Forward primer：NtNHX1-3F：GGATCCATGGCTTTCGACATTGGGATG；
Reverse primer：NtNHX1-3R：CTCGAGTTAATGATCACTTGGTTCAGT；
B, Leaf Explant of Nicotiana Tabacum L RNA is extracted, reverse transcription obtains the first chain cDNA；
C, using the first chain cDNA that reverse transcription obtains as template, PCR amplification is carried out with primer NtNHX1-3F/ NtNHX1-3R,
Recycling and purified pcr product；
D, purified product and carrier connect, and linked system is as follows with process：4 μ L purified products, 1 μ L salt solution, 1 μ L
PCR®-BluntⅡ-TOPO（Invitrogen）Mixing, 25 DEG C, water-bath 30min；The carrier connected is passed through heat-shock transformed
Escherichia coli DH5a adds the LB plate overnights training that the kanamycins containing 100mg/L is applied to after fluid nutrient medium shaken cultivation
It supports, picking colony carries out bacterium solution culture, plasmid extraction and PCR detections.Positive colony is sequenced in screening positive clone.
The third object of the present invention is achieved in that the gene NtNHX1-3 of the raising tobacco leaf potassium content is used
Application in the transfer-gen plant for obtaining the high potassium content of blade.
The present invention utilizes homologous clone method and round pcr, separation identification tobacco potassium transhipment by plant gene engineering technology
Body gene NtNHX1-3 sequence informations, and be overexpressed in tobacco cloud and mist 87 gene by Agrobacterium infestation method, pass through
The transgenosis T1 that qPCR detection screenings NtNHX1-3 is overexpressed is planted for plant, and potassium content inspection is carried out to the tobacco leaf of harvest
It surveys, obtains the tobacco plant that potassium content improves.The transfer-gen plant potassium content of acquisition is significantly improved than control.Illustrate tobacco
NtNHX1-3 genes have great application prospect in terms of the tobacco for cultivating high potassium content.
Description of the drawings
Fig. 1 is NtNHX1-3 gene PCR amplified production agarose gel electrophoresis figures, and NtNHX1-3 sizes are 1617bp.M is
DL2000 Maker, 1,2 is NtNHX1-3 amplified bands；
After Fig. 2 is NtNHX1-3 connection carriers TOPO, digestion identification is carried out using BamHI and XhoI, cut out 1617bp with
Two bar segments of 3500bp or so.M is DL2000 Maker, and 1,2,3 is digestion products；
After Fig. 3 is NtNHX1-3 connection entry clones carrier pENTR 2B, digestion identification is carried out using BamHI and XhoI, is cut out
Two bar segment of 1617bp and 3800bp.M is DL2000 Maker, and 1,2,3 is digestion products；
The digestion that Fig. 4 is plant expression vector pK2GW7-NtNHX1-3 is identified, is carried out digestion identification using BamHI and XhoI, is cut
Go out two bar segment of 1617bp and 11kb or so.M is DL2000 Maker, and 1,2,3 is digestion products；
Fig. 5 is to be overexpressed NtNHX1-3 gene transgenics T1 to analyze for strain qPCR, and PN3-4, PN2-5, PN3-9 are transgenic line
System, VC are to turn empty vector control；
Fig. 6 is transgene tobacco strain T1 for potassium content of tobacco leaf, and PN3-4, PN2-5, PN3-9 are transgenic line, and VC is to turn sky
Vehicle Control.
Specific implementation mode
With reference to embodiment and attached drawing, the present invention is further illustrated, but is not subject in any way to the present invention
Limitation, based on present invention teach that made by it is any transform or replace, all belong to the scope of protection of the present invention.
The nucleotide sequence such as sequence table SEQ of the gene NtNHX1-3 of the present invention for improving tobacco leaf potassium content
ID NO：Shown in 1.
The amino acid sequence such as SEQ ID NO of the gene NtNHX1-3 codings of the raising tobacco leaf potassium content：2 institutes
Show.
The cloning process of the gene NtNHX1-3 of the present invention for improving tobacco leaf potassium content, includes the following steps：
A, determinationNtNHX1-3Gene order；
Ncbi database, which is searched for, according to the protein sequence of rice NHX1 genes obtains homologous gene NtNHX1-3 sequences in tobacco, profit
With this sequence design gene cloning primer：
Forward primer：NtNHX1-3F：GGATCCATGGCTTTCGACATTGGGATG；
Reverse primer：NtNHX1-3R：CTCGAGTTAATGATCACTTGGTTCAGT；
B, Leaf Explant of Nicotiana Tabacum L RNA is extracted, reverse transcription obtains the first chain cDNA；
C, using the first chain cDNA that reverse transcription obtains as template, PCR amplification is carried out with primer NtNHX1-3F/ NtNHX1-3R,
Recycling and purified pcr product；
D, purified product and carrier connect, and linked system is as follows with process：4 μ L purified products, 1 μ L salt solution, 1 μ L
PCR®-BluntⅡ-TOPO（Invitrogen）Mixing, 25 DEG C, water-bath 30min；The carrier connected is passed through heat-shock transformed
Escherichia coli DH5a adds the LB plate overnights training that the kanamycins containing 100mg/L is applied to after fluid nutrient medium shaken cultivation
It supports, picking colony carries out bacterium solution culture, plasmid extraction and PCR detections.Positive colony is sequenced in screening positive clone.
The reaction system of PCR amplification is to select Phusion high-fidelity amplification enzyme reaction systems, system total volume in step C
50 μ L, including：The Phusion of 10 μ L, 10mM dNTP of 200ng cDNA, 5 × Phusion HF reaction buffers 1 μ L, 2U
High-Fidelity DNA Polymerase, 10 μM of each 1 μ L of forward and reverse primer, moisturizing to 50 μ L.
The reaction condition of PCR amplification is carried out on Mastercycler pro amplification instruments in step C, response procedures
For：98 DEG C, 30 seconds；98 DEG C, 7 seconds, 58 DEG C, 30 seconds, 72 DEG C, 30 seconds, 35 cycles；72 DEG C extend 7 minutes；
The over-express vector of the gene NtNHX1-3 of the present invention for improving tobacco leaf potassium content is pK2GW7-NtNHX1-
3。
The application of the gene NtNHX1-3 of the present invention for improving tobacco leaf potassium content is the raising Tobacco Leaf
The gene NtNHX1-3 of piece potassium content is used to obtain the application in the transfer-gen plant of the high potassium content of blade.
The method of transfer-gen plant for obtaining the high potassium content of blade includes the following steps：
A, over-express vector is built：
1, the NtNHX1-3 connection TOPO carriers cloned
（1）It using the cDNA of 87 blade of cloud and mist as template, is expanded using NtNHX1-3 gene specific primers, obtaining size is about
The genetic fragment of 1.6kb, purifying recycling；
（2）The NtNHX1-3 genetic fragments of recycling are subjected to TOPO clones, are turned after being connected to II-TOPO carriers of PCR-Blunt
Change escherichia coli DH5a competent cell, extraction plasmid carries out PCR detections, selects the plasmid that amplified production size is about 1.6kb
DNA is extracted, the carrier of structure is named as pTOPO-NtNHX1-3；
（3）Since the positive and negative trip primer of gene is respectively provided with the recognition site of BamH I, Xho I, the two enzymes is selected to confront
Grain DNA sample carries out double digestion detection, and digestion result generates two bar segments, and size is respectively 3.5kb and 1.6kb or so, explanation
Target fragment has been inserted into TOPO carriers；
2, the structure of plant over-express vector
A, the structure of entry clones pENTR 2B-NtNHX1-3
（1）BamH I/Xho I digestion pTOPO- NtNHX1-3 and pENTR 2B obtain target gene fragment NtNHX1-3 and load
Body pENTR 2B linearized fragments are attached, transformed competence colibacillus cell DH5a after glue recycling；
（2）Picking converts the clone after DH5a, extracts Plasmid DNA, BamH I/Xho I digestions, and digestion result is the load of 3.8kb
The segment of body segment and 1.6kb or so are that correctly clone, correct clone designation are pENTR 2B-NtNHX1-3；
B, plant expression vector is obtained by the reaction by LR
（1）Escherichia coli DH5a competence is converted after entry clones pENTR NtNHX1-3 and expression vector pK2GW7 LR reactions
Cell obtains plant expression vector pK2GW7-NtNHX1-3.It is identified using BamH I/Xho I digestions, correctly clone can be with
Cut out two bar segments about 1.6kb and 11kb.
Above-mentioned LR reaction systems：Build successful entry vector pENTR NtNHX1-3 (50-150ng) 1 ~ 7 μ L, 0.5
μ L Destination Vector, TE Buffer are supplemented to 8 μ L of total volume；Mixing, ice bath 2min are flicked 2 times；2 μ L are added
II enzyme Mix of LR CloneaseTM, are flicked, mixing, centrifugation, 25 DEG C of water-bath 1h；Then 1 μ L Proteinase K are added
It flicks, mixing, 37 DEG C of water-bath 10min；
B, the genetic transformation of tobacco：
（1）Expression vector converts Agrobacterium
Agrobacterium competent cell is taken out from -80 DEG C of refrigerators, places and recombinant expression carrier pK2GW7- is added after dissolving on ice
NtNHX1-3 4μL；Liquid nitrogen flash freezer 1 minute, is transferred to 37 DEG C of water-baths 5 minutes, then ice bath 2 minutes, and 1mL LB are added into mixture
Fluid nutrient medium, 28 DEG C, 220rpm cultivate 3 ~ 4 hours；Culture is coated on containing spectinomycin 100mg/L and rifampin
On the LB solid mediums of 25mg/L, 28 DEG C are inverted culture 2 ~ 3 days, it is seen that the Agrobacterium colonies containing purposeful carrier；
（2）Transformation of tobacco
A, picking contains the Agrobacterium colonies of destination carrier, in the flat lining outs of the LB containing spectinomycin and rifampin, 28 DEG C
Culture 2 ~ 3 days；Scraping scribing line bacterial plaque connects bacterium in the MS culture mediums containing spectinomycin and rifampin, 28 DEG C, 220rpm oscillations
Culture, 6 when bacterial concentration reaches OD=0.5 ~ 0.8,000rpm centrifuges 5 minutes enrichment thalline, abandons supernatant, then with 20mL liquid MS
Thalline is resuspended in culture medium, obtains the Agrobacterium suspension bacteria liquid containing destination carrier；
B, tobacco leaf is placed in 500mL wide-mouth bottles, appropriate 75% ethyl alcohol is added, rinse 1min；Ethyl alcohol is abandoned, is added 0.1%
HgCl2Solution sets shaken at room temperature 15 ~ 30 minutes on shaking table；Abandon HgCl2Solution, with aseptic water washing 6 times；
C, tobacco leaf is taken out, surface liquid is sucked with sterile blotting paper, using scissors by aseptic blade be cut into about 1cm ×
The tobacco leaf cut into pieces is put into the sterile MS fluid nutrient mediums suspension bacteria liquid containing destination carrier by the small pieces of 1cm, is stood
15~20min；Tobacco leaf is taken out, extra bacterium solution is sucked using aseptic filter paper, in containing 6-BA（0.02mg/L）,NAA
（2mg/L）MS culture mediums in 25 DEG C of light cultures two days；Then, tobacco leaf is transferred in differential medium, incision contacts training
Base is supported, culture is broken up under greenhouse experiment；Differential medium is to contain 6-BA（0.5mg/L）,NAA （0.1mg/L）, to block that mould
Element（100mg/L）, cephalosporin（500mg/L）MS culture mediums, every 2 ~ 3 weeks squamous subcultures 1 time, incision gradually grows callus
Tissue, final differentiation budding；
D, the long bud to 3 ~ 5cm is cut, is transferred to MS culture medium root inductions, take out the transfer-gen plant tap water after taking root
Culture medium is cleaned, is transplanted in the Nutrition Soil of sterilizing；
E, transfer-gen plant is through NPTII gene specific primers（NPTII-F:TCGGCTATGACTGGGCACAACAGA, NPTII-
R:AAGAAGGCGATAGAAGGCGATGCG) PCR verifications amplification, identifies transgenic positive plant.
0.1% HgCl2Solution preparation method is to weigh 0.1 gram of mercuric chloride, is dissolved with a little alcohol, and it is fixed to add water
Hold to 100 milliliters.
With specific embodiment, the present invention will be further described below：
Embodiment 1
The separation of NtNHX1-3 genes is cloned, including following steps：
1, NtNHX1-3 gene orders are determined；
According to the protein sequence of rice NHX1 genes（GenBank accession number Q68KI4.2）, search for ncbi database and obtain cigarette
Homologous gene NtNHX1-3 sequences in grass.Utilize this sequence design gene cloning primer：
Forward primer：NtNHX1-3F：GGATCCATGGCTTTCGACATTGGGATG；
Reverse primer：NtNHX1-3R：CTCGAGTTAATGATCACTTGGTTCAGT；
In order to build over-express vector, the primer restriction enzyme site in above-mentioned primer（Shown in underscore）BamH I and Xho I
2, RNAiso Plus are utilized（Takara）Leaf Explant of Nicotiana Tabacum L RNA is extracted, PrimeScript RT reagent Kit are utilized
with gDNA Eraser(Perfect Real Time)（Takara）Reverse transcription obtains the first chain cDNA；
3, using the first chain cDNA that reverse transcription obtains as template, with primer NtNHX1-3F/ NtNHX1-3R to carrying out PCR expansions
Increase, selection Phusion high-fidelity amplification enzyme reaction systems, 50 μ L of system total volume, including：200ng cDNA, 5 × Phusion
Phusion High-Fidelity the DNA Polymerase, 10 μ of 10 μ L, 10mM dNTP of HF reaction buffers 1 μ L, 2U
Each 1 μ L of forward and reverse primer of M, moisturizing to 50 μ L.PCR reactions carry out on Mastercycler pro amplification instruments, reaction interval
Sequence is：98 DEG C, 30 seconds；98 DEG C, 7 seconds, 58 DEG C, 30 seconds, 72 DEG C, 30 seconds, 35 cycles；72 DEG C extend 7 minutes；Recycling and purifying
PCR product；
4, purified product and carrier connect：Linked system is as follows with process：4 μ L purified products, 1 μ L salt solution, 1 μ
L PCR®-BluntⅡ-TOPO（Invitrogen）Mixing, 25 DEG C, water-bath 30min；The carrier connected is turned by heat shock
Change escherichia coli DH5a, adds the LB plate overnights training for being applied to the kanamycins containing 100mg/L after fluid nutrient medium shaken cultivation
It supports, picking colony carries out bacterium solution culture, plasmid extraction and PCR detections.Positive colony is sequenced in screening positive clone.
Culture medium prescription and preparation method described in step 4 are to weigh 8 ~ 12g of tryptone, yeast extract 5g,
NaCl 10g are dissolved in 1L distilled water, and 121 DEG C of sterilizing 25min are obtained.
Embodiment 2
Over-express vector is built
1, the NtNHX1-3 connection TOPO carriers cloned
（1）It using the cDNA of 87 blade of cloud and mist as template, is expanded using NtNHX1-3 gene specific primers, obtaining size is about
The genetic fragment of 1.6kb, purifying recycling；
（2）The NtNHX1-3 genetic fragments of recycling are subjected to TOPO clones, are turned after being connected to II-TOPO carriers of PCR-Blunt
Change escherichia coli DH5a competent cell, extraction plasmid carries out PCR detections, selects the plasmid that amplified production size is about 1.6kb
DNA is extracted, the carrier of structure is named as pTOPO-NtNHX1-3；
（3）Since the positive and negative trip primer of gene is respectively provided with the recognition site of BamH I, Xho I, the two enzymes is selected to confront
Grain DNA sample carries out double digestion detection, and digestion result generates two bar segments, and size is respectively 3.5kb and 1.6kb or so, explanation
Target fragment has been inserted into TOPO carriers；
2, the structure of plant over-express vector
A, the structure of entry clones pENTR 2B-NtNHX1-3
（1）BamH I/Xho I digestion pTOPO- NtNHX1-3 and pENTR 2B obtain target gene fragment NtNHX1-3 and load
Body pENTR 2B linearized fragments are attached, transformed competence colibacillus cell DH5a after glue recycling；
（2）Picking converts the clone after DH5a, extracts Plasmid DNA, BamH I/Xho I digestions, and digestion result is the load of 3.8kb
The segment of body segment and 1.6kb or so are that correctly clone, correct clone designation are pENTR 2B-NtNHX1-3；
B, plant expression vector is obtained by the reaction by LR
（1）Escherichia coli DH5a competence is converted after entry clones pENTR NtNHX1-3 and expression vector pK2GW7 LR reactions
Cell obtains plant expression vector pK2GW7-NtNHX1-3.PK2- NtNHX1-3 are identified using BamH I/Xho I digestions, just
True clone can cut out two bar segments about 1.6kb and 11kb.
Above-mentioned LR reaction systems：Build successful entry vector pENTR NtNHX1-3 (50-150ng) 1 ~ 7 μ L, 0.5
μ L Destination Vector, TE Buffer are supplemented to 8 μ L of total volume；Mixing, ice bath 2min are flicked 2 times；2 μ L are added
II enzyme Mix of LR CloneaseTM, are flicked, mixing, centrifugation, 25 DEG C of water-bath 1h；Then 1 μ L Proteinase K are added
It flicks, mixing, 37 DEG C of water-bath 10min.
Embodiment 3
The genetic transformation of tobacco
（1）Expression vector converts Agrobacterium
Agrobacterium competent cell is taken out from -80 DEG C of refrigerators, places and recombinant expression carrier pK2GW7- is added after dissolving on ice
NtNHX1-3 4μL；Liquid nitrogen flash freezer 1 minute, is transferred to 37 DEG C of water-baths 5 minutes, then ice bath 2 minutes, and 1mL LB are added into mixture
Fluid nutrient medium, 28 DEG C, 220rpm cultivate 3 ~ 4 hours；Culture is coated on containing spectinomycin 100mg/L and rifampin
On the LB solid mediums of 25mg/L, 28 DEG C are inverted culture 2 ~ 3 days, it is seen that the Agrobacterium colonies containing purposeful carrier；
（2）Transformation of tobacco
A, picking contains the Agrobacterium colonies of destination carrier, in the flat lining outs of the LB containing spectinomycin and rifampin, 28 DEG C
Culture 2 ~ 3 days；Scraping scribing line bacterial plaque connects bacterium in the MS culture mediums containing spectinomycin and rifampin, 28 DEG C, 220rpm oscillations
Culture, 6 when bacterial concentration reaches OD=0.5 ~ 0.8,000rpm centrifuges 5 minutes enrichment thalline, abandons supernatant, then with 20mL liquid MS
Thalline is resuspended in culture medium, obtains the Agrobacterium suspension bacteria liquid containing destination carrier；
B, tobacco leaf is placed in 500mL wide-mouth bottles, appropriate 75% ethyl alcohol is added, rinse 1min；Ethyl alcohol is abandoned, is added 0.1%
HgCl2Solution sets shaken at room temperature 15 ~ 30 minutes on shaking table；Abandon HgCl2Solution, with aseptic water washing 6 times；
C, tobacco leaf is taken out, surface liquid is sucked with sterile blotting paper, using scissors by aseptic blade be cut into about 1cm ×
The tobacco leaf cut into pieces is put into the sterile MS fluid nutrient mediums suspension bacteria liquid containing destination carrier by the small pieces of 1cm, is stood
15~20min；Tobacco leaf is taken out, extra bacterium solution is sucked using aseptic filter paper, in containing 6-BA（0.02mg/L）,NAA
（2mg/L）MS culture mediums in 25 DEG C of light cultures two days；Then, tobacco leaf is transferred in differential medium, incision contacts training
Base is supported, culture is broken up under greenhouse experiment；Differential medium is to contain 6-BA（0.5mg/L）,NAA （0.1mg/L）, to block that mould
Element（100mg/L）, cephalosporin（500mg/L）MS culture mediums, every 2 ~ 3 weeks squamous subcultures 1 time, incision gradually grows callus
Tissue, final differentiation budding；
D, the long bud to 3 ~ 5cm is cut, is transferred to MS culture medium root inductions, take out the transfer-gen plant tap water after taking root
Culture medium is cleaned, is transplanted in the Nutrition Soil of sterilizing；
E, transfer-gen plant is through NPTII gene specific primers（NPTII-F:TCGGCTATGACTGGGCACAACAGA, NPTII-
R:AAGAAGGCGATAGAAGGCGATGCG) PCR verifications amplification, identifies transgenic positive plant.
Embodiment 4
Transfer-gen plant is analyzed
Greenhouse pot culture is plantedNtNHX1-3Gene overexpression T1 is for strain PN3-4, PN3-5, PN3-9 and empty vector control VC.Often
Strain uses Special compound fertilizer for tobacco by 5g purity nitrogens（Dan ﹕ Lin ﹕ potassium=10 ﹕, 10 ﹕ 15）, point 5 applications.Squaring period, each strain selected 10
Strain takes middle leaf（9-12 leaves）, potassium content is detected after water-removing, as a result see the table below, and transgenic line PN3-4 potassium contents are than control
It improves 19.02%, PN3-5 potassium contents and improves 87.32% than control than control raising 117.58%, PN3-9 potassium contents.
Unit：%
Note：" * " indicates that above significant difference, " * * " indicate that the strain potassium content exists with VC to the strain potassium content in 0.05 level with VC
0.01 horizontal upper difference is extremely notable.
SEQUENCE LISTING
<110>Yunnan Academy of Tobacco Agricultural Science
<120>A kind of gene NtNHX1-3 improving tobacco leaf potassium content and its cloning process and application
<130> 2018
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 1617
<212> DNA
<213>The nucleotide sequence of NtNHX1-3
<400> 1
atggctttcg acattgggat gctgctggga aatattatga acaggttatc aacttctgat 60
catcaatcgg tggtgtcaat aaacttattt gttgcactta tctgcgcatg tatcgtggtc 120
ggtcatttgt tggaggaaaa tagatggatg aatgagtcca taactgccct cgtgattggt 180
ctttgcactg gagttgtcat tctactaata agtggtggga agaactctcg tattttagtg 240
ttcagcgaag atcttttctt catttacctt cttccaccga tcatttttaa tgctgggttc 300
caggtgaaaa agaaatcatt cttccgcaat ttcagcacca tcatgctttt tggagcagtt 360
ggcaccttga tatcattcat tatcatatca tttggtgcca ttggcatttt caagaaaatg 420
aacattggag accttgatat tggagattac cttgcaattg gagcaatctt ctctgcaacg 480
gattctgttt gcaccttaca agtgcttagt caggatgaca cacccttatt gtacagtcta 540
gtgtttgggg aaggtgttgt gaatgatgcc acatctgtgg ttctgttcaa tgctgtccag 600
aactttgact tatctcatat caacacaagc aaagctctgc aattagttgg aaactttctg 660
tacttgtttg cttcaagcac catcttaggg gttgttgctg gtctactgag cgcctatata 720
attaaaaaac tctactttgg aaggcactcc actgatcgtg aggttgctat aatgatactc 780
atggcttatc tgtcatacct gcttgctgaa ttattctatt taagtgcaat cctcactgtg 840
tttttctgtg ggatcgtgat gtctcactac acctggcata atgtgactga gagctcaaga 900
gtgaccacca agcacgcttt tgctacattg tcatttattg ctgagatatt catattcctt 960
tatgttggta tggatgcttt ggacatcgag aagtggaaat ttgtaagcga cagccctggt 1020
atatcagttc aggttagctc aatactgttg ggtcttgtta tggttggaag ggcagccttt 1080
gttttcccct tgtcattttt gtccaacttg accaagaagt ctccagagga gaagattggc 1140
tttaacaagc aaattgtaat atggtgggct ggacttatgc gaggtgctgt ttcagtggct 1200
ctggcttata atcagtttac cagaggaggt catactcagt tacgtggtaa tgcaataatg 1260
atcacgagta ccatcactgt tgtccttttc agcacagggg tgtttgggtt gatgacaaaa 1320
cctttaatta gatttatgct accctcacca aaacacttga ccagaatgat ctcttctgaa 1380
ccgacgaccc caaaatcctt cattgtgcca cttcttgaca gtgcacaaga ctcagaagct 1440
gatctgggcc aacatatacc ccgtcccaac agtttgcgga tgctcctatc aaccccatct 1500
cacactgtgc atcgttactg gagaaaattt gacaatgcgt tcatgcgtcc cgttttcggt 1560
ggacgaggtt ttgtaccttt tgttccagga tcaccgactg aaccaagtga tcattaa 1617
<210> 2
<211> 538
<212> PRT
<213>The amino acid sequence of NtNHX1-3 codings
<400> 2
Met Ala Phe Asp Ile Gly Met Leu Leu Gly Asn Ile Met Asn Arg Leu
1 5 10 15
Ser Thr Ser Asp His Gln Ser Val Val Ser Ile Asn Leu Phe Val Ala
20 25 30
Leu Ile Cys Ala Cys Ile Val Val Gly His Leu Leu Glu Glu Asn Arg
35 40 45
Trp Met Asn Glu Ser Ile Thr Ala Leu Val Ile Gly Leu Cys Thr Gly
50 55 60
Val Val Ile Leu Leu Ile Ser Gly Gly Lys Asn Ser Arg Ile Leu Val
65 70 75 80
Phe Ser Glu Asp Leu Phe Phe Ile Tyr Leu Leu Pro Pro Ile Ile Phe
85 90 95
Asn Ala Gly Phe Gln Val Lys Lys Lys Ser Phe Phe Arg Asn Phe Ser
100 105 110
Thr Ile Met Leu Phe Gly Ala Val Gly Thr Leu Ile Ser Phe Ile Ile
115 120 125
Ile Ser Phe Gly Ala Ile Gly Ile Phe Lys Lys Met Asn Ile Gly Asp
130 135 140
Leu Asp Ile Gly Asp Tyr Leu Ala Ile Gly Ala Ile Phe Ser Ala Thr
145 150 155 160
Asp Ser Val Cys Thr Leu Gln Val Leu Ser Gln Asp Asp Thr Pro Leu
165 170 175
Leu Tyr Ser Leu Val Phe Gly Glu Gly Val Val Asn Asp Ala Thr Ser
180 185 190
Val Val Leu Phe Asn Ala Val Gln Asn Phe Asp Leu Ser His Ile Asn
195 200 205
Thr Ser Lys Ala Leu Gln Leu Val Gly Asn Phe Leu Tyr Leu Phe Ala
210 215 220
Ser Ser Thr Ile Leu Gly Val Val Ala Gly Leu Leu Ser Ala Tyr Ile
225 230 235 240
Ile Lys Lys Leu Tyr Phe Gly Arg His Ser Thr Asp Arg Glu Val Ala
245 250 255
Ile Met Ile Leu Met Ala Tyr Leu Ser Tyr Leu Leu Ala Glu Leu Phe
260 265 270
Tyr Leu Ser Ala Ile Leu Thr Val Phe Phe Cys Gly Ile Val Met Ser
275 280 285
His Tyr Thr Trp His Asn Val Thr Glu Ser Ser Arg Val Thr Thr Lys
290 295 300
His Ala Phe Ala Thr Leu Ser Phe Ile Ala Glu Ile Phe Ile Phe Leu
305 310 315 320
Tyr Val Gly Met Asp Ala Leu Asp Ile Glu Lys Trp Lys Phe Val Ser
325 330 335
Asp Ser Pro Gly Ile Ser Val Gln Val Ser Ser Ile Leu Leu Gly Leu
340 345 350
Val Met Val Gly Arg Ala Ala Phe Val Phe Pro Leu Ser Phe Leu Ser
355 360 365
Asn Leu Thr Lys Lys Ser Pro Glu Glu Lys Ile Gly Phe Asn Lys Gln
370 375 380
Ile Val Ile Trp Trp Ala Gly Leu Met Arg Gly Ala Val Ser Val Ala
385 390 395 400
Leu Ala Tyr Asn Gln Phe Thr Arg Gly Gly His Thr Gln Leu Arg Gly
405 410 415
Asn Ala Ile Met Ile Thr Ser Thr Ile Thr Val Val Leu Phe Ser Thr
420 425 430
Gly Val Phe Gly Leu Met Thr Lys Pro Leu Ile Arg Phe Met Leu Pro
435 440 445
Ser Pro Lys His Leu Thr Arg Met Ile Ser Ser Glu Pro Thr Thr Pro
450 455 460
Lys Ser Phe Ile Val Pro Leu Leu Asp Ser Ala Gln Asp Ser Glu Ala
465 470 475 480
Asp Leu Gly Gln His Ile Pro Arg Pro Asn Ser Leu Arg Met Leu Leu
485 490 495
Ser Thr Pro Ser His Thr Val His Arg Tyr Trp Arg Lys Phe Asp Asn
500 505 510
Ala Phe Met Arg Pro Val Phe Gly Gly Arg Gly Phe Val Pro Phe Val
515 520 525
Pro Gly Ser Pro Thr Glu Pro Ser Asp His
530 535
<210> 3
<211> 27
<212> DNA
<213> NtNHX1-3F
<400> 3
ggatccatgg ctttcgacat tgggatg 27
<210> 4
<211> 27
<212> DNA
<213> NtNHX1-3R
<400> 4
ctcgagttaa tgatcacttg gttcagt 27
Claims (9)
1. a kind of gene NtNHX1-3 improving tobacco leaf potassium content is it is characterized in that the raising tobacco leaf potassium content
Gene NtNHX1-3 nucleotide sequence such as sequence table SEQ ID NO：Shown in 1.
2. the gene NtNHX1-3 according to claim 1 for improving tobacco leaf potassium content, it is characterised in that described carries
The amino acid sequence such as SEQ ID NO of the gene NtNHX1-3 codings of high tobacco leaf potassium content：Shown in 2.
3. a kind of cloning process of the gene NtNHX1-3 as claimed in claim 1 or 2 improving tobacco leaf potassium content, feature
It is to include the following steps：
A, determinationNtNHX1-3Gene order；
Ncbi database, which is searched for, according to the protein sequence of rice NHX1 genes obtains homologous gene NtNHX1-3 sequences in tobacco, profit
With this sequence design gene cloning primer：
Forward primer：NtNHX1-3F：GGATCCATGGCTTTCGACATTGGGATG；
Reverse primer：NtNHX1-3R：CTCGAGTTAATGATCACTTGGTTCAGT；
B, Leaf Explant of Nicotiana Tabacum L RNA is extracted, reverse transcription obtains the first chain cDNA；
C, using the first chain cDNA that reverse transcription obtains as template, PCR amplification is carried out with primer NtNHX1-3F/ NtNHX1-3R,
Recycling and purified pcr product；
D, purified product and carrier connect, and linked system is as follows with process：4 μ L purified products, 1 μ L salt solution, 1 μ L
PCR®-BluntⅡ-TOPO（Invitrogen）Mixing, 25 DEG C, water-bath 30min；The carrier connected is passed through heat-shock transformed
Escherichia coli DH5a adds the LB plate overnights training that the kanamycins containing 100mg/L is applied to after fluid nutrient medium shaken cultivation
It supports, picking colony carries out bacterium solution culture, and positive colony is sequenced in plasmid extraction and PCR detections, screening positive clone.
4. the cloning process of the gene NtNHX1-3 according to claim 3 for improving tobacco leaf potassium content, feature exist
The reaction system of PCR amplification is to select Phusion high-fidelity amplification enzyme reaction systems in step C, 50 μ L of system total volume, packet
It includes：The Phusion High- of 10 μ L, 10mM dNTP of 200ng cDNA, 5 × Phusion HF reaction buffers 1 μ L, 2U
Fidelity DNA Polymerase, 10 μM of each 1 μ L of forward and reverse primer, moisturizing to 50 μ L.
5. the cloning process of the gene NtNHX1-3 according to claim 3 for improving tobacco leaf potassium content, feature exist
The reaction condition of PCR amplification is carried out on Mastercycler pro amplification instruments in step C, and response procedures are：98 DEG C,
30 seconds；98 DEG C, 7 seconds, 58 DEG C, 30 seconds, 72 DEG C, 30 seconds, 35 cycles；72 DEG C extend 7 minutes.
6. a kind of over-express vector of the gene NtNHX1-3 as claimed in claim 1 or 2 improving tobacco leaf potassium content, special
Sign is that the over-express vector of the gene NtNHX1-3 of the raising tobacco leaf potassium content is pK2GW7-NtNHX1-3.
7. a kind of application of the gene NtNHX1-3 as claimed in claim 1 or 2 improving tobacco leaf potassium content, it is characterised in that
The gene NtNHX1-3 of the raising tobacco leaf potassium content is used to obtain answering in the transfer-gen plant of the high potassium content of blade
With.
8. the application of the gene NtNHX1-3 according to claim 7 for improving tobacco leaf potassium content, it is characterised in that use
Include the following steps in the method for the transfer-gen plant for obtaining the high potassium content of blade：
A, over-express vector is built：
1）The NtNHX1-3 connection TOPO carriers of clone
（1）It using the cDNA of 87 blade of cloud and mist as template, is expanded using NtNHX1-3 gene specific primers, obtaining size is about
The genetic fragment of 1.6kb, purifying recycling；
（2）The NtNHX1-3 genetic fragments of recycling are subjected to TOPO clones, are turned after being connected to II-TOPO carriers of PCR-Blunt
Change escherichia coli DH5a competent cell, extraction plasmid carries out PCR detections, selects the plasmid that amplified production size is about 1.6kb
DNA is extracted, the carrier of structure is named as pTOPO-NtNHX1-3；
（3）Since the positive and negative trip primer of gene is respectively provided with the recognition site of BamH I, Xho I, the two enzymes is selected to confront
Grain DNA sample carries out double digestion detection, and digestion result generates two bar segments, and size is respectively 3.5kb and 1.6kb or so, explanation
Target fragment has been inserted into TOPO carriers；
2）The structure of plant over-express vector
A, the structure of entry clones pENTR 2B-NtNHX1-3
（1）BamH I/Xho I digestion pTOPO- NtNHX1-3 and pENTR 2B obtain target gene fragment NtNHX1-3 and load
Body pENTR 2B linearized fragments are attached, transformed competence colibacillus cell DH5a after glue recycling；
（2）Picking converts the clone after DH5a, extracts Plasmid DNA, BamH I/Xho I digestions, and digestion result is the load of 3.8kb
The segment of body segment and 1.6kb or so are that correctly clone, correct clone designation are pENTR 2B-NtNHX1-3；
B, plant expression vector is obtained by the reaction by LR
（1）Escherichia coli DH5a competence is converted after entry clones pENTR NtNHX1-3 and expression vector pK2GW7 LR reactions
Cell obtains plant expression vector pK2GW7-NtNHX1-3；It is identified using BamH I/Xho I digestions, correctly clone can be with
Cut out two bar segments about 1.6kb and 11kb；
Above-mentioned LR reaction systems：Build successful entry vector pENTR NtNHX1-3 (50-150ng) 1 ~ 7 μ L, 0.5 μ L
Destination Vector, TE Buffer are supplemented to 8 μ L of total volume；Mixing, ice bath 2min are flicked 2 times；2 μ L LR are added
II enzyme Mix of CloneaseTM, are flicked, mixing, centrifugation, 25 DEG C of water-bath 1h；Then it is light that 1 μ L Proteinase K are added
Bullet, mixing, 37 DEG C of water-bath 10min；
B, the genetic transformation of tobacco：
（1）Expression vector converts Agrobacterium
Agrobacterium competent cell is taken out from -80 DEG C of refrigerators, places and recombinant expression carrier pK2GW7- is added after dissolving on ice
NtNHX1-3 4μL；Liquid nitrogen flash freezer 1 minute, is transferred to 37 DEG C of water-baths 5 minutes, then ice bath 2 minutes, and 1mL LB are added into mixture
Fluid nutrient medium, 28 DEG C, 220rpm cultivate 3 ~ 4 hours；Culture is coated on containing spectinomycin 100mg/L and rifampin
On the LB solid mediums of 25mg/L, 28 DEG C are inverted culture 2 ~ 3 days, it is seen that the Agrobacterium colonies containing purposeful carrier；
（2）Transformation of tobacco
A, picking contains the Agrobacterium colonies of destination carrier, in the flat lining outs of the LB containing spectinomycin and rifampin, 28 DEG C
Culture 2 ~ 3 days；Scraping scribing line bacterial plaque connects bacterium in the MS culture mediums containing spectinomycin and rifampin, 28 DEG C, 220rpm oscillations
Culture, 6 when bacterial concentration reaches OD=0.5 ~ 0.8,000rpm centrifuges 5 minutes enrichment thalline, abandons supernatant, then with 20mL liquid MS
Thalline is resuspended in culture medium, obtains the Agrobacterium suspension bacteria liquid containing destination carrier；
B, tobacco leaf is placed in 500mL wide-mouth bottles, appropriate 75% ethyl alcohol is added, rinse 1min；Ethyl alcohol is abandoned, is added 0.1%
HgCl2Solution sets shaken at room temperature 15 ~ 30 minutes on shaking table；Abandon HgCl2Solution, with aseptic water washing 6 times；
C, tobacco leaf is taken out, surface liquid is sucked with sterile blotting paper, using scissors by aseptic blade be cut into about 1cm ×
The tobacco leaf cut into pieces is put into the sterile MS fluid nutrient mediums suspension bacteria liquid containing destination carrier by the small pieces of 1cm, is stood
15~20min；Tobacco leaf is taken out, extra bacterium solution is sucked using aseptic filter paper, in containing 6-BA（0.02mg/L）,NAA
（2mg/L）MS culture mediums in 25 DEG C of light cultures two days；Then, tobacco leaf is transferred in differential medium, incision contacts training
Base is supported, culture is broken up under greenhouse experiment；Differential medium is to contain 6-BA（0.5mg/L）,NAA （0.1mg/L）, to block that mould
Element（100mg/L）, cephalosporin（500mg/L）MS culture mediums, every 2 ~ 3 weeks squamous subcultures 1 time, incision gradually grows callus
Tissue, final differentiation budding；
D, the long bud to 3 ~ 5cm is cut, is transferred to MS culture medium root inductions, take out the transfer-gen plant tap water after taking root
Culture medium is cleaned, is transplanted in the Nutrition Soil of sterilizing；
E, transfer-gen plant is through NPTII gene specific primers（NPTII-F:TCGGCTATGACTGGGCACAACAGA, NPTII-
R:AAGAAGGCGATAGAAGGCGATGCG) PCR verifications amplification, identifies transgenic positive plant.
9. the application of the gene NtNHX1-3 according to claim 8 for improving tobacco leaf potassium content, it is characterised in that institute
0.1% HgCl stated2Solution preparation method is to weigh 0.1 gram of mercuric chloride, is dissolved with alcohol, adds water and be settled to 100 milliliters.
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109337918A (en) * | 2018-11-13 | 2019-02-15 | 云南省烟草农业科学研究院 | A kind of tobacco protein kinase gene NtCIPK1 and its cloning process and application |
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| CN109468329A (en) * | 2018-11-13 | 2019-03-15 | 云南省烟草农业科学研究院 | A kind of tobacco outward rectification potassium-channel gene NtSKOR1 and its cloning process and application |
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Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1884539A (en) * | 2006-06-12 | 2006-12-27 | 黑龙江省烟草科学研究所 | Use of Arabidopsis thaliana proton pump gene AtNHX1 for promoting plant rootage absorbing potassium capability |
| US20070107082A1 (en) * | 2003-06-03 | 2007-05-10 | Cropdesign N.V. | Transgenic monocotyledonous plants overexpressing a nhx protein and having improved growth characteristics and a method for making the same |
| CN101906433A (en) * | 2009-06-05 | 2010-12-08 | 中国科学院植物研究所 | Application of Na+/H+ reverse transport protein and protein-coding gene thereof in culturing disease-resistant transgenic plant |
| CN102964436A (en) * | 2012-11-12 | 2013-03-13 | 中国科学院植物研究所 | Plant salt-resistant disease-resistant related protein NtNHX1, and coding gene and application thereof |
| CN103602688A (en) * | 2013-11-28 | 2014-02-26 | 南京农业大学 | Helianthus tuberosus L. Na<+>/H<+> reverse transport protein genes HtNHX1 and HtNHX2 and use thereof |
| CN103937906A (en) * | 2014-05-12 | 2014-07-23 | 蒋丹 | Qualitative standard sample of transgenic tobacco NHX1 as well as preparation and valuing methods of qualitative standard sample |
| WO2015035532A1 (en) * | 2013-09-11 | 2015-03-19 | Institute Of Botany | Transgenic plants |
| CN106613924A (en) * | 2016-12-26 | 2017-05-10 | 上海烟草集团有限责任公司 | Method for screening and identifying potassium-rich tobacco |
| WO2017217508A1 (en) * | 2016-06-17 | 2017-12-21 | 積水化学工業株式会社 | Method for improving salt tolerance of plant |
| CN107828795A (en) * | 2017-11-09 | 2018-03-23 | 云南省烟草农业科学研究院 | A kind of gene NtNHX1 2 for improving tobacco leaf potassium content and its cloning process and application |
| CN109913446A (en) * | 2019-03-22 | 2019-06-21 | 罗银 | A kind of clone of tobacco NtNHX2 gene and under different adverse circumstances expression pattern analysis method |
-
2018
- 2018-06-28 CN CN201810683684.8A patent/CN108753795A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20070107082A1 (en) * | 2003-06-03 | 2007-05-10 | Cropdesign N.V. | Transgenic monocotyledonous plants overexpressing a nhx protein and having improved growth characteristics and a method for making the same |
| CN1884539A (en) * | 2006-06-12 | 2006-12-27 | 黑龙江省烟草科学研究所 | Use of Arabidopsis thaliana proton pump gene AtNHX1 for promoting plant rootage absorbing potassium capability |
| CN101906433A (en) * | 2009-06-05 | 2010-12-08 | 中国科学院植物研究所 | Application of Na+/H+ reverse transport protein and protein-coding gene thereof in culturing disease-resistant transgenic plant |
| CN102964436A (en) * | 2012-11-12 | 2013-03-13 | 中国科学院植物研究所 | Plant salt-resistant disease-resistant related protein NtNHX1, and coding gene and application thereof |
| WO2015035532A1 (en) * | 2013-09-11 | 2015-03-19 | Institute Of Botany | Transgenic plants |
| CN103602688A (en) * | 2013-11-28 | 2014-02-26 | 南京农业大学 | Helianthus tuberosus L. Na<+>/H<+> reverse transport protein genes HtNHX1 and HtNHX2 and use thereof |
| CN103937906A (en) * | 2014-05-12 | 2014-07-23 | 蒋丹 | Qualitative standard sample of transgenic tobacco NHX1 as well as preparation and valuing methods of qualitative standard sample |
| WO2017217508A1 (en) * | 2016-06-17 | 2017-12-21 | 積水化学工業株式会社 | Method for improving salt tolerance of plant |
| CN106613924A (en) * | 2016-12-26 | 2017-05-10 | 上海烟草集团有限责任公司 | Method for screening and identifying potassium-rich tobacco |
| CN107828795A (en) * | 2017-11-09 | 2018-03-23 | 云南省烟草农业科学研究院 | A kind of gene NtNHX1 2 for improving tobacco leaf potassium content and its cloning process and application |
| CN109913446A (en) * | 2019-03-22 | 2019-06-21 | 罗银 | A kind of clone of tobacco NtNHX2 gene and under different adverse circumstances expression pattern analysis method |
Non-Patent Citations (14)
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109337918A (en) * | 2018-11-13 | 2019-02-15 | 云南省烟草农业科学研究院 | A kind of tobacco protein kinase gene NtCIPK1 and its cloning process and application |
| CN109468334A (en) * | 2018-11-13 | 2019-03-15 | 云南省烟草农业科学研究院 | A kind of tobacco protein kinase gene NtCIPK25-1 and its cloning process and application |
| CN109468329A (en) * | 2018-11-13 | 2019-03-15 | 云南省烟草农业科学研究院 | A kind of tobacco outward rectification potassium-channel gene NtSKOR1 and its cloning process and application |
| CN109468329B (en) * | 2018-11-13 | 2022-04-08 | 云南省烟草农业科学研究院 | Tobacco outward rectifying potassium ion channel gene NtSKOR1, and cloning method and application thereof |
| CN109553667A (en) * | 2018-11-14 | 2019-04-02 | 贵州省烟草科学研究院 | Tobacco KUP2 gene and application |
| CN109553667B (en) * | 2018-11-14 | 2021-08-31 | 贵州省烟草科学研究院 | Tobacco KUP2 gene and application thereof |
| CN110305884A (en) * | 2019-08-05 | 2019-10-08 | 云南省烟草农业科学研究院 | A kind of gene NtAOS1 improving tobacco leaf jasmine acid content and its cloning process and application |
| CN111138519A (en) * | 2020-01-06 | 2020-05-12 | 河南农业大学 | Over-expression gene capable of improving potassium content of tobacco, and coding product and application thereof |
| CN111138519B (en) * | 2020-01-06 | 2022-03-18 | 河南农业大学 | Over-expression gene capable of improving potassium content of tobacco, and coding product and application thereof |
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Application publication date: 20181106 |