CN109468329A - A kind of tobacco outward rectification potassium-channel gene NtSKOR1 and its cloning process and application - Google Patents
A kind of tobacco outward rectification potassium-channel gene NtSKOR1 and its cloning process and application Download PDFInfo
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- CN109468329A CN109468329A CN201811347121.8A CN201811347121A CN109468329A CN 109468329 A CN109468329 A CN 109468329A CN 201811347121 A CN201811347121 A CN 201811347121A CN 109468329 A CN109468329 A CN 109468329A
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- SYSWVVCYSXBVJG-RHYQMDGZSA-N Val-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)N)O SYSWVVCYSXBVJG-RHYQMDGZSA-N 0.000 description 1
- BGXVHVMJZCSOCA-AVGNSLFASA-N Val-Pro-Lys Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)O)N BGXVHVMJZCSOCA-AVGNSLFASA-N 0.000 description 1
- HWNYVQMOLCYHEA-IHRRRGAJSA-N Val-Ser-Tyr Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N HWNYVQMOLCYHEA-IHRRRGAJSA-N 0.000 description 1
- JXCOEPXCBVCTRD-JYJNAYRXSA-N Val-Tyr-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N JXCOEPXCBVCTRD-JYJNAYRXSA-N 0.000 description 1
- RTJPAGFXOWEBAI-SRVKXCTJSA-N Val-Val-Arg Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N RTJPAGFXOWEBAI-SRVKXCTJSA-N 0.000 description 1
- NLNCNKIVJPEFBC-DLOVCJGASA-N Val-Val-Glu Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O NLNCNKIVJPEFBC-DLOVCJGASA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000008186 active pharmaceutical agent Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000003698 anagen phase Effects 0.000 description 1
- 108010007483 arginyl-leucyl-tyrosyl-glutamic acid Proteins 0.000 description 1
- 125000003118 aryl group Chemical group 0.000 description 1
- 108010077245 asparaginyl-proline Proteins 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 108010016616 cysteinylglycine Proteins 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000003205 fragrance Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000012215 gene cloning Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000010362 genome editing Methods 0.000 description 1
- 108010080575 glutamyl-aspartyl-alanine Proteins 0.000 description 1
- 108010049041 glutamylalanine Proteins 0.000 description 1
- 108010010147 glycylglutamine Proteins 0.000 description 1
- 108010081551 glycylphenylalanine Proteins 0.000 description 1
- 108010025306 histidylleucine Proteins 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 239000002932 luster Substances 0.000 description 1
- 108010003700 lysyl aspartic acid Proteins 0.000 description 1
- 108010044348 lysyl-glutamyl-aspartic acid Proteins 0.000 description 1
- 239000011707 mineral Substances 0.000 description 1
- 235000010755 mineral Nutrition 0.000 description 1
- 239000003595 mist Substances 0.000 description 1
- 238000002887 multiple sequence alignment Methods 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
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- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
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Abstract
The invention discloses a kind of tobacco outward rectification potassium-channel genesNtSKOR1And its cloning process and application;The tobacco outward rectification potassium-channel geneNtSKOR1Nucleotide sequence is as shown in SEQ ID NO:1;The amino acid sequence of albumen is encoded as shown in SEQ ID NO:2;Cloning process includes tobacco leaf cDNA synthesis: extracting tobacco leaf total serum IgE, reverse transcription obtains the first chain cDNA;NtSKOR1The PCR amplification of gene: using tobacco leaf cDNA as template, according toNtSKOR1Gene order design primer carries out PCR amplification, recycling and purifying pcr amplification product, and is sequenced.Using for the tobacco outward rectification potassium-channel geneNtSKOR1Application in the transgenic tobacco plant of regulation tobacco potassium content.The present invention is using CRISPR/CAS9 technology to clone'sNtSKOR1Gene has carried out Function Identification.TobaccoNtSKOR1The clone identification of gene provides new gene target for regulation tobacco potassium content.
Description
Technical field
The invention belongs to genetic engineering technology fields, and in particular to a kind of tobacco outward rectification potassium-channel geneNtSKOR1And its cloning process and application.
Background technique
Potassium is a kind of one of mineral nutrient element necessary to plant growth, is the most abundant monovalence sun of plant in-vivo content
Ion.Plant absorption potassium is mainly to be carried out by root, and the form of absorption is K+.The mode of plant absorption potassium is divided into two kinds:
Active absorption and Passive intake.Active absorption requires the expenditure of energy, but the rate of active absorption is very high.The K in the soil liquid+It is dense
When spending higher, K+Absorption be mainly Passive intake.In Potassium In Plants with K+Form exist.Root absorb K+ be easy to by
It is transported to overground part, is also easy to be transferred to other positions from a position in plant, and can be weighed in plant
It is multiple to utilize.In the insufficient situation of Potassium In Plants, potassium is preferentially assigned to raw one long vigorous tender tissue, in organ.
Potassium-channel mainly includes Shaker potassium-channel, the channel TPK and the channel Kir-like in plant.SKOR
Belong to Shaker potassium-channel family,SKOR It is mainly expressed in root, works as K +It is rapidly absorbed into root, during SKOR passes through
Column makes it be transferred to xylem and transports overground part bud to xylem sap, this may be only to potassium in xylem sap point
Secrete the Shaker K to work + Channel.In addition, SKOR is also expressed in xylem and pollen.Some researches show that, SKOR with
GORK can be carried out physics interaction and form functional different poly- outward rectification channel.
Potassium is considered as the Quality Element of tobacco, and potassium content is one of the important indicator for evaluating quality of tobacco superiority and inferiority.Tobacco leaf
Potassium content improves the institutional framework that can improve tobacco leaf, it is fine and smooth to make tobacco leaf structure, and can also improve tobacco leaf appearance luster, makes cigarette
Ye Chengshen crocus, fragrance foot, jealous good, high resilience and toughness, fillibility enhancing.In addition potassium can also enhance tobacco leaf sugar
Class, pigment, aromatic substance dynamic accumulation.It yet there are no the report of SKOR potassium-channel gene in tobacco, clone tobacco
Potassium-channel geneSKOR, exchange tobacco control grass potassium content, cultivation high quality tobacco leaf is of great significance.
Summary of the invention
The first object of the present invention is to provide a kind of tobacco outward rectification potassium-channel geneNtSKOR1;Second mesh
Be the tobacco outward rectification potassium-channel gene is providedNtSKOR1Cloning process;Third is designed to provide
The tobacco outward rectification potassium-channel geneNtSKOR1Application.
The first object of the present invention is achieved in that the tobacco outward rectification potassium-channel geneNtSKOR1
Nucleotide sequence as shown in SEQ ID NO:1.
The second object of the present invention be achieved in that the following steps are included:
A, tobacco leaf cDNA is synthesized: extracting tobacco root tissue RNA, reverse transcription obtains the first chain cDNA;
B、NtSKOR1The PCR amplification of gene: using tobacco leaf cDNA as template, according toNtSKOR1Gene order design primer,
Carry out PCR amplification, recycling and purifying pcr amplification product;
C, purified product is linked with carrier, and linked system is as follows with process: 4 μ L purified products, 1 μ L salt solution, 1 μ L
II-TOPO(Invitrogen of PCR-Blunt) it mixes, 25 DEG C, water-bath 30min;The carrier connected is passed through heat-shock transformed
Escherichia coli DH5a adds the LB plate overnight 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 plasmid extracts and positive colony is sequenced in PCR detection, screening positive clone.
The third object of the present invention is achieved in that the tobacco outward rectification potassium-channel geneNtSKOR1
Application in the transgenic tobacco plant for obtaining low potassium ion content.
The present invention also provides a kind of tobacco outward rectification potassium-channel genesNtSKOR1Recombinant vector.
The present invention also provides provide a kind of tobacco outward rectification potassium-channel geneNtSKOR1Expression cassette.
The present invention also provides a kind of tobacco outward rectification potassium-channel genesNtSKOR1Transgenic cell line.
The present invention also provides a kind of tobacco outward rectification potassium-channel genesNtSKOR1Recombinant bacterium.
The present invention also provides a kind of using CRSIPR/CAS9 editing technique to tobaccoNtSKOR1Gene carries out Function Identification
Method, specifically includes the following steps:
(1) CRISPR/CAS9 carrier is constructed
A, basisNtSKOR1 Genome sequence designs CRISPR/CAS9 target site (PAM), i.e.,
GAGAGTAGCAGAGGAAGTACTGG, the target site are located at First Exon.
B, target site design of primers
Target site primer is synthesized according to the target site of A design:
P1:5 '-ATTGGAGAGTAGCAGAGGAAGTAC -3 ',
P2:5 '-AAACGTACTTCCTCTGCTACTCTC -3 ';
Wherein P1 gray area is 20 nucleotide before target site TGG, and the grey area P2 is the reverse mutual of P1 gray area
Complementary series.
C, in the detection primer of target site two sides design editing material,
SdF:5 '-TCCATCACACGAACTCTCCA -3 ',
SdR:5 '-GAGAGCAAGAGAGAGAGGAGT -3 ', amplification length 475bp.
D, dsDNA is prepared
The primer that synthesis is designed in step B is formed into complementary DNA oligo by annealing, the specific steps are as follows:
50 μ L of reaction system, including 20 20 μ L, 10 × Annealing buffer of μ L, SdR of SdF, 5 μ L, sterilize 5 μ of distilled water
L.Cycle of annealing are as follows: 95 DEG C, 5min;90 DEG C, 1 min;80 DEG C, 1 min;70 DEG C, 1 min;60 DEG C, 1 min;50 DEG C, 1min;
40 DEG C, 1 min;30 DEG C, 1 min;20 DEG C, 1 min;10 DEG C, 1 min.
E, digestion pHSE401 carrier, and be attached with dsDNA prepared by step D.
Digestion, 50 μ L of digestion system, comprising: plasmid 5 μ L, 10 × buffer are carried out to pHSE401 carrier using BsaI enzyme
52 μ L of μ L, BsaI, sterilize 38 μ L of distilled water.37 DEG C of digestion 1h.
Electrophoresis detection analysis is carried out to digestion products after digestion, it is seen that two bands of 1200bp and 11520bp, recycling
The digestion products of 11520bp are spare;
The large fragment digestion products recycled are attached with dsDNA prepared by step D using T4 DNA ligase, when connection
20 μ L systems carry out: recycled 3 μ L of carrier digestion products, and anneal formed 10 μ L, T4 DNA buffer of dsDNA product, 2 μ
1 μ L of L, T4 DNA ligase, sterilize 4 μ L of distilled water.16 DEG C of connections overnight;
F, sequence verification
Connection product in step E is converted into Escherichia coli, screening positive clone (pHSE401 carrier resistance is kan) simultaneously carries out bacterium
PCR detection is fallen,
When bacterium colony PCR is detected, the primer design:
U6-26p F:5 '-TGTCCCAGGATTAGAATGATTAGGC -3 ';
P2:5 '-AAACGTACTTCCTCTGCTACTCTC -3 ';
Further progress sequencing analysis after correct positive colony bacterial strain culture expands, sequencing when institute are verified to bacterium colony PCR detection
With primer are as follows:
U6-26p-F: 5’- TGTCCCAGGATTAGAATGATTAGGC -3’。
Sequencing result is analyzed, correctly clone (pHSE401-SKOR1) saves backup for selection building.
(2) Agrobacterium-mediated Transformation
Agrobacterium competent cell (C58C1) is taken out from -80 DEG C of refrigerators, places and carrier pHSE401- is added after dissolving on ice
SKOR1 4μL;Liquid nitrogen flash freezer 1 minute, 37 DEG C of water-baths 5 minutes, then ice bath 2 minutes are transferred to, 1mL LB liquid is added into mixture
Body culture medium, 28 DEG C, 220rpm culture 3 ~ 4 hours;Culture is coated on 100mg/L and rifampin 25mg/L containing kanamycin
LB solid medium on, 28 DEG C of inversions are cultivated 2 ~ 3 days, it is seen that the Agrobacterium colonies containing destination carrier.
(3) Transformation of tobacco
A, picking contains the Agrobacterium colonies of destination carrier, in the flat lining out of the LB containing that mycin and rifampin, 28 DEG C of trainings
It supports 2 ~ 3 days;Scraping scribing line bacterial plaque connects bacterium in the MS culture medium containing that mycin and rifampin, and 28 DEG C, 220rpm shake culture,
Bacterial concentration is infected when reaching OD=0.5 ~ 0.8;
B, tobacco leaf is placed in 500mL wide-mouth bottle, appropriate 75% ethyl alcohol is added, rinse 1min;Ethyl alcohol is abandoned, is added 0.1%
HgCl2 solution sets shaken at room temperature 15 ~ 30 minutes on shaking table;Solution is abandoned, with aseptic water washing 6 times;
C, blade is taken out, washes away surface liquid with sterile blotting paper, aseptic blade is taken to be cut into the small pieces of 1cm × 1cm with scissors,
The tobacco leaf cut into pieces is put into the sterile MS fluid nutrient medium suspension bacteria liquid containing destination carrier, 15 ~ 20min is stood;
Tobacco leaf is taken out, sucks extra bacterium solution, the MS culture of Yu Hanyou 6-BA (0.02mg/L), NAA (2mg/L) with aseptic filter paper
25 DEG C dark culture two days in base;Tobacco leaf is transferred in differential medium, incision contacts culture medium, differential medium be containing
6-BA (0.5mg/L), NAA (0.1mg/L), hygromycin (20mg/L), cephalosporin (500mg/L) MS culture medium, every 2 ~ 3
Zhou Jidai is primary, and incision gradually forms callus, finally differentiation budding;
D, the long bud to 3 ~ 5cm is cut, is transferred to MS culture medium root induction, the transgenic plant after taking root is by root media
Middle taking-up is cleaned culture medium with tap water, is transplanted in the Nutrition Soil of sterilizing.
(4) sequencing screening editor material
T0 is grown 1 week or so for transgenic seedling, is chosen 20 plants of tobacco seedlings and is taken blade and utilize DNeasy Plant Mini Kit
(QIAGEN) DNA is extracted, is expanded using the primer SdF/SdR designed in step (1) C, amplified production utilizes just after purification
It is sequenced to primer.Sequencing result is analyzed, editor's material (Fig. 4) of one plant of insertion, 1 bases G is obtained.Plant editor's material
In T1 generation, edits homozygous single plant sowing by sequencing screening and obtains T2 generation.
(5) material potassium content is edited
Greenhouse pot culture plantation editor homozygosis material NtSKOR1-CP and control.Every plant uses Special compound fertilizer for tobacco by 5g purity nitrogen
(Dan ﹕ Lin ﹕ potassium=10 ﹕, 10 ﹕ 15), point 5 applications.Squaring period takes upper leaf (16-19 leaves), middle leaf (9-11 leaves), lower part
Leaf (3-5 leaves), detects potassium content after water-removing.As a result see that Fig. 5, editor's three position blade potassium contents of material are more significant than compareing
It reduces, showsNtSKOR1Transport of the potassium ion to overground part is affected after gene knockout.
The present invention obtains a tobacco outward rectification potassium-channel gene using Homology-based cloning from tobaccoNtSKOR1, utilize CRISPR/CAS9 editing technique pairNtSKOR1Carry out functional verification, the results showed thatNtSKOR1Gene has
Potassium ion is transported to the function of overground part from root.In tobaccoNtSKOR1The identification of gene has not been reported, the mirror of the gene
It is set to and new gene is provided by the expression regulation tobacco potassium content of controlling gene.
Detailed description of the invention
Fig. 1 is to be expanded using primer pair NtSKOR1F/NtSKOR1RNtSKOR1The Ago-Gel of gene C DS product
Electrophoretogram, amplified production size is about 2466bp, M:DL2000,1:NtSKOR1 CDS PCR product;
Fig. 2 isNtSKOR1Tissue-specific expression analysis as a result,NtSKOR1Gene is mainly expressed in root;
Fig. 3 is that NtSKOR1 albumen is compared with arabidopsis AtSKOR protein sequence, and wherein S1-S6 is 6 transmembrane regions of prediction, P
For hole formation area, ANK is anchorin area;
Fig. 4 is that CRISPR/CAS9 is editedNtSKOR1Genetic material editor's target site sequencing result, wherein PAM is editor's target position
Point, Wild are wild-type sequence, and CP is editor's material sequence, and NtSKOR1-CP material is inserted into a bases G;
Fig. 5 isNtSKOR1Gene editing material is compared with control top, middle part, lower tobacco leaf potassium content.NtSKOR1-CP is to compile
Material is collected, CK is control.
Specific embodiment
Below 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 it is made it is any transform or replace, all belong to the scope of protection of the present invention.
Tobacco outward rectification potassium-channel gene of the present inventionNtSKOR1Nucleotide sequence such as SEQ ID NO:
Shown in 1.
The tobacco outward rectification potassium-channel geneNtSKOR1The amino acid sequence of coding such as SEQ ID NO:2
It is shown.
Tobacco outward rectification potassium-channel gene of the present inventionNtSKOR1Cloning process, comprising the following steps:
A, tobacco leaf cDNA is synthesized: extracting tobacco root tissue RNA, reverse transcription obtains the first chain cDNA;
B、NtSKOR1The PCR amplification of gene: using tobacco leaf cDNA as template, according toNtSKOR1Gene order design primer,
Carry out PCR amplification, recycling and purifying pcr amplification product;
C, purified product is linked with carrier, and linked system is as follows with process: 4 μ L purified products, 1 μ L salt solution, 1 μ L
II-TOPO(Invitrogen of PCR-Blunt) it mixes, 25 DEG C, water-bath 30min;The carrier connected is passed through heat-shock transformed
Escherichia coli DH5a adds the LB plate overnight 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 plasmid extracts and positive colony is sequenced in PCR detection, screening positive clone.
Primer described in step B are as follows:
NtSKOR1F:5 '-ATGACGAGAGTAGCAGAGGA -3 '
NtSKOR1R:5 '-TCAAGTTGATTGATCATTGATC-3 '.
PCR reaction system described in step B is to select Phusion high-fidelity amplification enzyme reaction system, system total volume
50 μ L, comprising: the Phusion of 10 μ L, 10mM dNTP of 200ng cDNA, 5 × Phusion HF reaction buffer 1 μ L, 2U
High-Fidelity DNA Polymerase, each 1 μ L of 10 μM of forward and reverse primer, moisturizing to 50 μ L.
The reaction condition of PCR amplification described in step B is carried out on Mastercycler pro amplification instrument, reaction
Program are as follows: 98 DEG C, 30 seconds;98 DEG C, 7 seconds;58 DEG C, 30 seconds;72 DEG C, 60 seconds;35 circulations;72 DEG C extend 7 minutes.
Tobacco outward rectification potassium-channel gene of the present inventionNtSKOR1Application be that the described tobacco extroversion is whole
Flow potassium-channel geneNtSKOR1Application in the transgenic tobacco plant for obtaining low potassium ion content.
Contain tobacco outward rectification potassium-channel gene of any of claims 1 or 2NtSKOR1Recombinant vector.
Contain tobacco outward rectification potassium-channel gene of any of claims 1 or 2NtSKOR1Expression cassette.
Contain tobacco outward rectification potassium-channel gene of any of claims 1 or 2NtSKOR1Transgenic cell line
Or recombinant bacterium.
Case is embodied, the present invention will be further described below:
Embodiment 1
Tobacco potassium ion outward rectification geneNtSKOR1Clone
A, according to arabidopsisAtSKORProtein sequence (NP_186934.1) the search ncbi database of gene obtains homologous in tobacco
GeneNtSKOR1Sequence utilizes this sequence design gene cloning primer:
Forward primer: NtSKOR1F:5 '-ATGACGAGAGTAGCAGAGGA-3 '
Reverse primer: NtSKOR1R:5 '-TCAAGTTGATTGATCATTGATC-3 '
B, tobacco root tissue 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 NtSKOR1F/ NtSKOR1R,
PCR product recycles after agarose gel electrophoresis separates and purifies (Fig. 1);
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
II-TOPO(Invitrogen of PCR-Blunt) it mixes, 25 DEG C, water-bath 30min;The carrier connected is passed through heat-shock transformed
Escherichia coli DH5a adds the LB plate overnight 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 plasmid extracts and positive colony is sequenced in PCR detection, screening positive clone.
The reaction system of PCR amplification is to select Phusion high-fidelity amplification enzyme reaction system, system total volume in step C
50 μ L, comprising: the Phusion of 10 μ L, 10mM dNTP of 200ng cDNA, 5 × Phusion HF reaction buffer 1 μ L, 2U
High-Fidelity DNA Polymerase, each 1 μ L of 10 μM of forward and reverse primer, moisturizing to 50 μ L.
The reaction condition of PCR amplification is carried out on Mastercycler pro amplification instrument in step C, response procedures
Are as follows: 98 DEG C, 30 seconds;98 DEG C, 7 seconds;58℃;30 seconds, 72 DEG C;60 seconds, 35 circulations;72 DEG C extend 7 minutes;
Embodiment 2
TobaccoNtSKOR1Tissue-specific expression analysis
A, growing and cultivation tobacco cloud and mist 87, prosperous growth phase extract root, stem, blade RNA, and reverse transcription obtains the first chain cDNA.
B, basisNtSKOR1Gene order designs qRT-PCR primer
QF:GTCAAGTTGTAACTCGAGTCCAC,
QR:GGAAATATCTCCGAATGAGCTG.
Using tobacco Actin gene as internal reference, Actin-F:CTGAGGTCCTTTTCCAACCA and Actin-
RTACCCGGGAACATGGTAGAG.Quantitative fluorescent PCR is carried out using root, stem, leaf cDNA as template, is reacted in Roche
It is carried out on LightCycler 480 using 480 SYBR Green I master of LightCycler, 20 μ L systems contain 10
480 SYBR Green I master(2 × of μ L LightCycler), positive each 1 μ L(10 μm ol/L of anti-primer), cDNA 1
μ L(reverse transcription product dilutes 4 times), 7 μ L sterile purified waters.Response procedures are as follows: 95 DEG C of 5 min of initial denaturation;95 DEG C of denaturation
20 s, 60 DEG C of annealing 15 s, 72 DEG C of 15 s of extension;45 circulations.Fluorescent quantitative PCR result uses 2-△△CtMethod calculatesNtSKOR1The relative expression quantity of gene.Each processing sets 3 biology and repeats, by Excel Software on Drawing histogram (Fig. 2).Knot
Fruit showsNtSKOR1Mainly expressed in tobacco root.
Embodiment 3
TobaccoNtSKOR1Gene coded protein guards domain analysis
NtSKOR1 albumen and arabidopsis AtSKOR albumen are subjected to Multiple Sequence Alignment (Fig. 3).NtSKOR1 is protected with SKOR albumen
Structural domain is kept, such as transmembrane region S1-S6, hole formation area P, the conserved domains such as anchorin area ANK, and NtSKOR1 are in these knots
The area Gou Yu and AtSKOR homology are higher.
Embodiment 4
Using CRSIPR/CAS9 editing technique to tobaccoNtSKOR1Gene carries out Function Identification
(1) CRISPR/CAS9 carrier is constructed
A, basisNtSKOR1 Genome sequence designs CRISPR/CAS9 target site (PAM), i.e.,
GAGAGTAGCAGAGGAAGTACTGG, the target site are located at First Exon.
B, target site design of primers
Target site primer is synthesized according to the target site of A design:
P1:5 '-ATTGGAGAGTAGCAGAGGAAGTAC -3 ',
P2:5 '-AAACGTACTTCCTCTGCTACTCTC -3 ';
Wherein P1 gray area is 20 nucleotide before target site TGG, and the grey area P2 is the reverse mutual of P1 gray area
Complementary series.
C, in the detection primer of target site two sides design editing material,
SdF:5 '-TCCATCACACGAACTCTCCA -3 ',
SdR:5 '-GAGAGCAAGAGAGAGAGGAGT -3 ', amplification length 475bp.
D, dsDNA is prepared
The primer that synthesis is designed in step B is formed into complementary DNA oligo by annealing, the specific steps are as follows:
50 μ L of reaction system, including 20 20 μ L, 10 × Annealing buffer of μ L, SdR of SdF, 5 μ L, sterilize 5 μ of distilled water
L.Cycle of annealing are as follows: 95 DEG C, 5min;90 DEG C, 1 min;80 DEG C, 1 min;70 DEG C, 1 min;60 DEG C, 1 min;50 DEG C, 1min;
40 DEG C, 1 min;30 DEG C, 1 min;20 DEG C, 1 min;10 DEG C, 1 min.
E, digestion pHSE401 carrier, and be attached with dsDNA prepared by step D.
Digestion, 50 μ L of digestion system, comprising: plasmid 5 μ L, 10 × buffer are carried out to pHSE401 carrier using BsaI enzyme
52 μ L of μ L, BsaI, sterilize 38 μ L of distilled water.37 DEG C of digestion 1h.
Electrophoresis detection analysis is carried out to digestion products after digestion, it is seen that two bands of 1200bp and 11520bp, recycling
The digestion products of 11520bp are spare;
The large fragment digestion products recycled are attached with dsDNA prepared by step D using T4 DNA ligase, when connection
20 μ L systems carry out: recycled 3 μ L of carrier digestion products, and anneal formed 10 μ L, T4 DNA buffer of dsDNA product, 2 μ
1 μ L of L, T4 DNA ligase, sterilize 4 μ L of distilled water.16 DEG C of connections overnight;
F, sequence verification
Connection product in step E is converted into Escherichia coli, screening positive clone (pHSE401 carrier resistance is kan) simultaneously carries out bacterium
PCR detection is fallen,
When bacterium colony PCR is detected, the primer design:
U6-26p F:5 '-TGTCCCAGGATTAGAATGATTAGGC -3 ';
P2:5 '-AAACGTACTTCCTCTGCTACTCTC -3 ';
Further progress sequencing analysis after correct positive colony bacterial strain culture expands, sequencing when institute are verified to bacterium colony PCR detection
With primer are as follows:
U6-26p-F: 5’- TGTCCCAGGATTAGAATGATTAGGC -3’。
Sequencing result is analyzed, correctly clone (pHSE401-SKOR1) saves backup for selection building.
(2) Agrobacterium-mediated Transformation
Agrobacterium competent cell (C58C1) is taken out from -80 DEG C of refrigerators, places and carrier pHSE401- is added after dissolving on ice
SKOR1 4μL;Liquid nitrogen flash freezer 1 minute, 37 DEG C of water-baths 5 minutes, then ice bath 2 minutes are transferred to, 1mL LB liquid is added into mixture
Body culture medium, 28 DEG C, 220rpm culture 3 ~ 4 hours;Culture is coated on 100mg/L and rifampin 25mg/L containing kanamycin
LB solid medium on, 28 DEG C of inversions are cultivated 2 ~ 3 days, it is seen that the Agrobacterium colonies containing destination carrier.
(3) Transformation of tobacco
A, picking contains the Agrobacterium colonies of destination carrier, in the flat lining out of the LB containing that mycin and rifampin, 28 DEG C of trainings
It supports 2 ~ 3 days;Scraping scribing line bacterial plaque connects bacterium in the MS culture medium containing that mycin and rifampin, and 28 DEG C, 220rpm shake culture,
Bacterial concentration is infected when reaching OD=0.5 ~ 0.8;
B, tobacco leaf is placed in 500mL wide-mouth bottle, appropriate 75% ethyl alcohol is added, rinse 1min;Ethyl alcohol is abandoned, is added 0.1%
HgCl2 solution sets shaken at room temperature 15 ~ 30 minutes on shaking table;Solution is abandoned, with aseptic water washing 6 times;
C, blade is taken out, washes away surface liquid with sterile blotting paper, aseptic blade is taken to be cut into the small pieces of 1cm × 1cm with scissors,
The tobacco leaf cut into pieces is put into the sterile MS fluid nutrient medium suspension bacteria liquid containing destination carrier, 15 ~ 20min is stood;
Tobacco leaf is taken out, sucks extra bacterium solution, the MS culture of Yu Hanyou 6-BA (0.02mg/L), NAA (2mg/L) with aseptic filter paper
25 DEG C dark culture two days in base;Tobacco leaf is transferred in differential medium, incision contacts culture medium, differential medium be containing
6-BA (0.5mg/L), NAA (0.1mg/L), hygromycin (20mg/L), cephalosporin (500mg/L) MS culture medium, every 2 ~ 3
Zhou Jidai is primary, and incision gradually forms callus, finally differentiation budding;
D, the long bud to 3 ~ 5cm is cut, is transferred to MS culture medium root induction, the transgenic plant after taking root is by root media
Middle taking-up is cleaned culture medium with tap water, is transplanted in the Nutrition Soil of sterilizing.
(4) sequencing screening editor material
T0 is grown 1 week or so for transgenic seedling, is chosen 20 plants of tobacco seedlings and is taken blade and utilize DNeasy Plant Mini Kit
(QIAGEN) DNA is extracted, is expanded using the primer SdF/SdR designed in step (1) C, amplified production utilizes just after purification
It is sequenced to primer.Sequencing result is analyzed, editor's material (Fig. 4) of one plant of insertion, 1 bases G is obtained.Plant editor's material
In T1 generation, edits homozygous single plant sowing by sequencing screening and obtains T2 generation.
(5) material potassium content is edited
Greenhouse pot culture plantation editor homozygosis material NtSKOR1-CP and control.Every plant uses Special compound fertilizer for tobacco by 5g purity nitrogen
(Dan ﹕ Lin ﹕ potassium=10 ﹕, 10 ﹕ 15), point 5 applications.Squaring period takes upper leaf (16-19 leaves), middle leaf (9-11 leaves), lower part
Leaf (3-5 leaves), detects potassium content after water-removing.As a result see that Fig. 5, editor's three position blade potassium contents of material are more significant than compareing
It reduces, showsNtSKOR1Transport of the potassium ion to overground part is affected after gene knockout.
SEQUENCE LISTING
<110>Yunnan Academy of Tobacco Agricultural Science
<120>a kind of tobacco outward rectification potassium-channel gene NtCIPK1 and its cloning process and application
<130> 2018
<160> 8
<170> PatentIn version 3.3
<210> 1
<211> 1365
<212> DNA
<213>NtCIPK1 nucleotide sequence
<400> 1
atggtattga tacagcaaga agaagaaata agaagtgaaa gaggaaagaa gggaatgcga 60
gttgggaaat acgaacttgg aaaaacttta ggagaaggta attttggcaa agttaagtac 120
gcaaaacaca aagattctgg ccaatctttt gctatcaaaa ttttggagaa aaatcgaatt 180
caagatctta gaatcaccga tcagataaag agggagatta aaaccttaaa agtcctcaag 240
catccaaatg tggtcagatt atacgaggtc ttagcaagca aaaccaagat ttacatggtg 300
ctggaatatg taaatggtgg tgaattattt gacagaattg cttctgaagg aaaactagca 360
gaaacacaag gcagaaaact ctttcaacaa ttaattgatg gtgttagtta ttgccatgac 420
aaaggtgtct tccatagaga cctcaagcta gagaatgtcc tcattgatgc agaaaaaaac 480
ataaagatta cagattttgg actaagtgca ttacctcaac acttaaggga tgacggcttg 540
ttgcatacaa catgtggtag ccccaactac gttgctcctg aaattctttc taacagagga 600
tacgatggcg cgacatcaga tacatggtca tgtggagtca tcttatatgt cattctcact 660
ggttttttac cctttgatga tagaaatctt gcagttcttt atcaaaagat ttttaagggg 720
gatgcaccag taccaaaatg gttatctcaa ggagcaaaga atcttataaa gaggattctt 780
gatccaaatc cgcatactcg cataacaatg gcagagatta aagaggatga gtggttcaaa 840
caagactata ctcctgctaa tcctgatgaa tatgaagatt ttgaagatga tcatgcatcc 900
tcagatgatg aagtgttgac agtacatgaa gcaccacttg atacagaaag agatccagaa 960
tcaccttctg ttatcaataa tgcctttcag ctaattggga tgtcctcatg ttttgatctt 1020
tctggatttt ttgaaaatga ggatgcctct gagaggaaga tcagattcac atctaatctc 1080
tctccaaaag aattgctaga gaggattgag catttagcag tgcaaatggg atttcaagtc 1140
cagaaaaaac ctggaaagtt gaaagtattg ctagaacaca aaggtcaaaa aactcaagcc 1200
agtctttcaa tagtagcaga ggtttttgag attagcacat ccttgtatgt tgtagagtta 1260
caaaaatctt caggggattc tacggtatat agagagttat gcaataggtt atcaaatgaa 1320
ttgggtgtcc agcaaagtca agagctcttg cccaccaaat tatga 1365
<210> 2
<211> 454
<212> PRT
<213>NtCIPK1 amino acid sequence
<400> 2
Met Val Leu Ile Gln Gln Glu Glu Glu Ile Arg Ser Glu Arg Gly Lys
1 5 10 15
Lys Gly Met Arg Val Gly Lys Tyr Glu Leu Gly Lys Thr Leu Gly Glu
20 25 30
Gly Asn Phe Gly Lys Val Lys Tyr Ala Lys His Lys Asp Ser Gly Gln
35 40 45
Ser Phe Ala Ile Lys Ile Leu Glu Lys Asn Arg Ile Gln Asp Leu Arg
50 55 60
Ile Thr Asp Gln Ile Lys Arg Glu Ile Lys Thr Leu Lys Val Leu Lys
65 70 75 80
His Pro Asn Val Val Arg Leu Tyr Glu Val Leu Ala Ser Lys Thr Lys
85 90 95
Ile Tyr Met Val Leu Glu Tyr Val Asn Gly Gly Glu Leu Phe Asp Arg
100 105 110
Ile Ala Ser Glu Gly Lys Leu Ala Glu Thr Gln Gly Arg Lys Leu Phe
115 120 125
Gln Gln Leu Ile Asp Gly Val Ser Tyr Cys His Asp Lys Gly Val Phe
130 135 140
His Arg Asp Leu Lys Leu Glu Asn Val Leu Ile Asp Ala Glu Lys Asn
145 150 155 160
Ile Lys Ile Thr Asp Phe Gly Leu Ser Ala Leu Pro Gln His Leu Arg
165 170 175
Asp Asp Gly Leu Leu His Thr Thr Cys Gly Ser Pro Asn Tyr Val Ala
180 185 190
Pro Glu Ile Leu Ser Asn Arg Gly Tyr Asp Gly Ala Thr Ser Asp Thr
195 200 205
Trp Ser Cys Gly Val Ile Leu Tyr Val Ile Leu Thr Gly Phe Leu Pro
210 215 220
Phe Asp Asp Arg Asn Leu Ala Val Leu Tyr Gln Lys Ile Phe Lys Gly
225 230 235 240
Asp Ala Pro Val Pro Lys Trp Leu Ser Gln Gly Ala Lys Asn Leu Ile
245 250 255
Lys Arg Ile Leu Asp Pro Asn Pro His Thr Arg Ile Thr Met Ala Glu
260 265 270
Ile Lys Glu Asp Glu Trp Phe Lys Gln Asp Tyr Thr Pro Ala Asn Pro
275 280 285
Asp Glu Tyr Glu Asp Phe Glu Asp Asp His Ala Ser Ser Asp Asp Glu
290 295 300
Val Leu Thr Val His Glu Ala Pro Leu Asp Thr Glu Arg Asp Pro Glu
305 310 315 320
Ser Pro Ser Val Ile Asn Asn Ala Phe Gln Leu Ile Gly Met Ser Ser
325 330 335
Cys Phe Asp Leu Ser Gly Phe Phe Glu Asn Glu Asp Ala Ser Glu Arg
340 345 350
Lys Ile Arg Phe Thr Ser Asn Leu Ser Pro Lys Glu Leu Leu Glu Arg
355 360 365
Ile Glu His Leu Ala Val Gln Met Gly Phe Gln Val Gln Lys Lys Pro
370 375 380
Gly Lys Leu Lys Val Leu Leu Glu His Lys Gly Gln Lys Thr Gln Ala
385 390 395 400
Ser Leu Ser Ile Val Ala Glu Val Phe Glu Ile Ser Thr Ser Leu Tyr
405 410 415
Val Val Glu Leu Gln Lys Ser Ser Gly Asp Ser Thr Val Tyr Arg Glu
420 425 430
Leu Cys Asn Arg Leu Ser Asn Glu Leu Gly Val Gln Gln Ser Gln Glu
435 440 445
Leu Leu Pro Thr Lys Leu
450
<210> 3
<211> 22
<212> DNA
<213> NtCIPK1F
<400> 3
atggtattga tacagcaaga ag 22
<210> 4
<211> 24
<212> DNA
<213> NtCIPK1R
<400> 4
tcataatttg gtgggcaaga gctc 24
<210> 5
<211> 24
<212> DNA
<213> P1
<400> 5
attgggaaag aagggaatgc gagt 24
<210> 6
<211> 24
<212> DNA
<213> P2
<400> 6
aaacactcgc attcccttct ttcc 24
<210> 7
<211> 20
<212> DNA
<213> CIPK1dF
<400> 7
atctaagcag cggattgacg 20
<210> 8
<211> 21
<212> DNA
<213> CIPK1dR
<400> 8
agcagagaga gagagacctg a 21
Claims (10)
1. a kind of tobacco outward rectification potassium-channel geneNtSKOR1, it is characterised in that the tobacco outward rectification potassium from
Subchannel geneNtSKOR1Nucleotide sequence as shown in SEQ ID NO:1.
2. tobacco outward rectification potassium-channel gene according to claim 1NtSKOR1, it is characterised in that the cigarette
Careless outward rectification potassium-channel geneNtSKOR1The amino acid sequence of coding is as shown in SEQ ID NO:2.
3. a kind of tobacco outward rectification potassium-channel gene of any of claims 1 or 2NtSKOR1Cloning process, it is special
Sign be the following steps are included:
A, tobacco leaf cDNA is synthesized: extracting tobacco root tissue RNA, reverse transcription obtains the first chain cDNA;
B、NtSKOR1The PCR amplification of gene: using tobacco leaf cDNA as template, according toNtSKOR1Gene order design primer,
Carry out PCR amplification, recycling and purifying pcr amplification product;
C, purified product is linked with carrier, and linked system is as follows with process: 4 μ L purified products, 1 μ L salt solution, 1 μ L
II-TOPO(Invitrogen of PCR-Blunt) it mixes, 25 DEG C, water-bath 30min;The carrier connected is passed through heat-shock transformed
Escherichia coli DH5a adds the LB plate overnight 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 plasmid extracts and positive colony is sequenced in PCR detection, screening positive clone.
4. tobacco outward rectification potassium-channel gene according to claim 3NtSKOR1Cloning process, feature exists
Primer described in step B are as follows:
NtSKOR1F:5 '-ATGACGAGAGTAGCAGAGGA -3 '
NtSKOR1R:5 '-TCAAGTTGATTGATCATTGATC-3 '.
5. tobacco outward rectification potassium-channel gene according to claim 3NtSKOR1Cloning process, feature exists
PCR reaction system described in step B are as follows: 50 μ L of total volume, comprising: 200ng cDNA, 5 × Phusion HF reaction buffering
The Phusion High-Fidelity DNA Polymerase of 10 μ L, 10mM dNTP of liquid 1 μ L, 2U, 10 μM forward and reverse
Each 1 μ L of primer, moisturizing to 50 μ L.
6. tobacco outward rectification potassium-channel gene according to claim 3NtSKOR1Cloning process, feature exists
The response procedures of PCR amplification described in step B are as follows: 98 DEG C, 30 seconds;98 DEG C, 7 seconds;58 DEG C, 30 seconds;72 DEG C, 60 seconds;35
A circulation;72 DEG C extend 7 minutes.
7. a kind of tobacco outward rectification potassium-channel gene of any of claims 1 or 2NtSKOR1Application, feature exists
In the tobacco outward rectification potassium-channel geneNtSKOR1In the transgenic tobacco plant of regulation tobacco potassium content
In application.
8. one kind contains tobacco outward rectification potassium-channel gene of any of claims 1 or 2NtSKOR1Recombinant vector.
9. one kind contains tobacco outward rectification potassium-channel gene of any of claims 1 or 2NtSKOR1Expression cassette.
10. one kind contains tobacco outward rectification potassium-channel gene of any of claims 1 or 2NtSKOR1Transgenosis it is thin
Born of the same parents system or recombinant bacterium.
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Cited By (4)
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CN108374014A (en) * | 2018-02-08 | 2018-08-07 | 云南省烟草农业科学研究院 | A kind of gene NtTPKa improving tobacco leaf potassium content and its cloning process and application |
CN111454340A (en) * | 2020-04-02 | 2020-07-28 | 北京市农林科学院 | Elytrigia elongata external rectification potassium channel protein and coding gene and application thereof |
CN112143738A (en) * | 2020-09-30 | 2020-12-29 | 云南省烟草农业科学研究院 | Tobacco receptor protein gene and cloning method and application thereof |
CN118240834A (en) * | 2024-03-07 | 2024-06-25 | 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) | Tobacco potassium channel gene NtSKOR1B and its application in improving tobacco salt stress resistance |
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