CN106350524A - Tobacco axillary bud growth regulation gene NtBRC1 and cloning method and application thereof - Google Patents
Tobacco axillary bud growth regulation gene NtBRC1 and cloning method and application thereof Download PDFInfo
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- CN106350524A CN106350524A CN201610960510.2A CN201610960510A CN106350524A CN 106350524 A CN106350524 A CN 106350524A CN 201610960510 A CN201610960510 A CN 201610960510A CN 106350524 A CN106350524 A CN 106350524A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
- C12N15/8267—Seed dormancy, germination or sprouting
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- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/686—Polymerase chain reaction [PCR]
Abstract
The invention discloses a tobacco axillary bud growth regulation gene NtBRC1 and a cloning method and application thereof. The tobacco axillary bud growth regulation gene NtBRC1 has a nucleotide sequence shown as SEQ ID: No.1 and an encoded amino acid sequence shown as SEQ ID: No.2. The invention also discloses a cloning method of the tobacco axillary bud growth regulation gene NtBRC1. The method comprises the following specific steps: A, determining a gene sequence of NtBRC1; B, extracting tobacco RNA, and performing reverse transcription to obtain a first strand of cDNA; C, designing and synthesizing a specific primer according to the gene sequence of NtBRC1, taking cDNA as a template, and performing PCR amplification; D, recovering and purifying the PCR product; E, purifying the product and vector connection, and transforming competent cells; and F, screening positive clone, and performing PCR amplification and sequencing on the positive clone. By regulating the expression of the tobacco axillary bud growth regulation gene NtBRC1, the development length of the topped tobacco axillary buds can be reduced, so that the usage of a tobacco bud inhibitor is expected to be reduced, and gene resources are provided for producing green high-quality tobacco leaves and reducing the labor cost.
Description
Technical field
The invention belongs to gene engineering field and in particular to a kind of tobacco axillary bud adjusting and controlling growth gene ntbrc1 and its
Cloning process and application.
Background technology
Nicotiana tabacum L. (nicotiana tabacum) it is important industrial crops, it is Solanaceae annual herb plant.Pinch bud picking
It is important production technology measures in high-quality tobacco planting process.Nicotiana tabacum L. must be pinched in certain growth period, and beats
Substantial amounts of lateral bud can be germinated behind top.Lateral bud expends nutrition, can reduce the quality and yield of Nicotiana tabacum L. it is therefore necessary to lateral bud of erasing.People
Work bud picking labor intensive and material resources, improve leaf tobacco production cost.Application After Topping chemical agent can suppress axillary bud growth, reduces
Leaf tobacco production labor intensity.But chemical agent needs cost, and environmental pollution can be caused.With invention to find to produce after pinching
The related gene of lateral bud (axillary bud) is breach, creates the Nicotiana tabacum L. of no lateral bud, few lateral bud or little lateral bud by biotechnology, then has
May fundamentally or significantly solve the above problems, thus realize the aspects such as tobacco growing amount, quality of tobacco, environmental protection all having
Larger lifting.
brc1It is the transcription factor protein gene of a tcp family.Its function in controlling branch growth is a large amount of
Report,brc1The arabidopsiss of gene mutation increase than wild type lotus throne；Semen Pisi sativibrc1Gene mutation posterior division increases.We are to cigarette
Grassntbrc1Gene has been also carried out extensive theoretical research and application practice, findsntbrc1There is after tobbaco promotion lateral bud
The function that (axillary bud) grows.Controlling bud growth promoter aspect on rear side of tobbaco to have production by plant gene engineering technology should
Use significance.
Some well-known tobacco companies and research institution just adopt biotechnology and technique for gene engineering to Nicotiana tabacum L. no in the world
The few axillary bud kind of axillary bud is studied, and to improve cigarette quality, reduces the administration to chemical agent after tobbaco, to reduce cigarette
Leaf productive labor intensity, chemical agent cost and environmental pollution.At present, China it is also proposed the exploitation meter of no axillary bud High Quality Tobacco
Draw, be that the few axillary bud kind research of Nicotiana tabacum L. no axillary bud provides unlimited opportunity.Depth with the few axillary bud kind research of Nicotiana tabacum L. no axillary bud
Enter, China's tobacco axillary bud will be made to control and reach a new stage, be that health and the sustainable development of China's Nicotiana tabacum L. lays the foundation.Cause
This, a kind of candidate gene of exploitation utilization animal nutrition initiative no few axillary bud kind of axillary bud, reduces cigarette by genetic manipulation
Careless axillary bud is very important.
Content of the invention
The first object of the present invention is to provide a kind of tobacco axillary bud adjusting and controlling growth gene ntbrc1；Second purpose is to carry
Cloning process for described tobacco axillary bud adjusting and controlling growth gene ntbrc1；3rd purpose is to provide described tobacco axillary bud life
The application of long controlling gene ntbrc1.
The first object of the present invention is achieved in that the nucleotide of described tobacco axillary bud adjusting and controlling growth gene ntbrc1
Sequence is as shown in seq id no:1.
The second object of the present invention is achieved in that and comprises the following steps:
A, determinationntbrc1Gene order；
(1) obtain Nicotiana tabacum L. using tomato dna sibrc1b search tobacco gene group data basentbrc1Nucleotide sequence, utilize
This sequential design gene cloning primer:
Forward primer: 5 '-atgtatccgccaagcaacag -3 '；
Reverse primer: 5 '-gctattgaaatcctaaaaaat -3 '；
B, extraction Nicotiana tabacum L. root tissue rna, reverse transcription obtains the first chain cdna；
C, basisntbrc1Gene order design synthesis specific primer, the first chain cdna being obtained using reverse transcription as template,
Carry out pcr amplification, from phusion high-fidelity amplification enzyme reaction system, system cumulative volume 50 μ l, comprising: 200ng cdna, 5
× phusion hf reaction buffer, the phusion high-fidelity dna polymerase of 10mm dntp, 2u,
Each 1 μm of forward and reverse primer, pcr reaction is carried out on mastercycler pro amplification instrument, and response procedures are: 98 DEG C, 30 seconds；
98 DEG C, 7 seconds, 55 DEG C, 30 seconds, 72 DEG C, 30 seconds, 35 circulations；72 DEG C extend 7 minutes；Reclaim and purification pcr product；
D, purified product are connected with carrier, are connected with topo carrier by test kit reaction, linked system is as follows with process: 4 μ l
Purified product+1 μ l salt salution+1 μ l pcr-blunt-topo mixes, 25 DEG C, water-bath 30min；By connected
Carrier is by heat-shock transformed entrance escherichia coli, plus is coated onto the lb containing 100mg/l kanamycin after fluid medium concussion and cultivate
Plate overnight is cultivated, and after growing bacterium, picking colony is cultivated, and takes 2ml bacterium solution to extract plasmid, carry out plasmid pcr after culture
Detection, screening positive clone, positive colony is sequenced.
The third object of the present invention is achieved in that described tobacco axillary bud adjusting and controlling growth genentbrc1Obtaining fall
Low pinch after Nicotiana tabacum L. produce axillary bud the transfer-gen plant of growth length in application.
The present invention obtains a tobacco axillary bud adjusting and controlling growth base using Homology-based cloning from Nicotiana tabacum L.ntbrc1, concrete step
Suddenly it is: using using tomato dna sibrc1b(genbank accession number: hm597230) search tobacco gene group data base
(https: //solgenomics.net/) obtains Nicotiana tabacum L.ntbrc1Nucleotide sequence.Drawn using this sequential design gene cloning
Thing；Carry out pcr reaction using this sequence information design gene specific primer, obtain purpose product；Purpose product is sequenced, obtainsntbrc1Gene order；Obtained using agrobcterium-mediated transformationntbrc1Overexpression (oe) plant of gene, rightntbrc1Carry out functional verification, result showsntbrc1Gene has the function of controlling Nicotiana tabacum L. lateral bud growth promoter.ntbrc1Base
The discovery of cause, is the expression by controlling gene, controls Nicotiana tabacum L. lateral bud growth promoter, provides base for producing green high quality Nicotiana tabacum L.
Because of resource.
Brief description
Fig. 1 is using the amplification of primer pair ntbrc1f/ntbrc1rntbrc1Genetic results, amplified production 987bp；
Fig. 2 is topo-ntbrc1Carrier pcr identifies, to for ntbrc1f/ntbrc1r, amplified production is 987 bp to the primer；
Fig. 3 is topo-ntbrc1Carrier enzyme action is identified, utilizesbamHi andxhoI double digestion, it is possible to obtain 1000bp about
Purpose band；
Fig. 4 isntbrc1Gene overexpression (oe) carrier target fragment pcr amplification, the primer is to for ntbrc1-oe-f
/ ntbrc1-oe-r, amplified production is 999 bp；
Fig. 5 is the digestion verification of overexpression (oe) carrier,bamHi andxhoAfter i double digestion, can by 1000bp about purpose
Band cuts；
Fig. 6 isntbrc1Overexpression (oe) tobacco axillary bud growth length, vector control(vc) for turning empty vector control, its
Remaining for transgenic line.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is further illustrated, but never in any form to the present invention in addition
Limit, based on present invention teach that any conversion of being made or replacement, belong to protection scope of the present invention.
The nucleotide sequence of tobacco axillary bud adjusting and controlling growth gene ntbrc1 of the present invention is as shown in seq id no:1.
The aminoacid sequence of described tobacco axillary bud adjusting and controlling growth gene ntbrc1 coding is as shown in seq id no:2.
The cloning process of tobacco axillary bud adjusting and controlling growth gene ntbrc1 of the present invention is it is characterised in that include following walking
Rapid:
A, determinationntbrc1Gene order；
(1) obtain Nicotiana tabacum L. using tomato dna sibrc1b search tobacco gene group data basentbrc1Nucleotide sequence, utilize
This sequential design gene cloning primer:
Forward primer: 5 '-atgtatccgccaagcaacag -3 '；
Reverse primer: 5 '-gctattgaaatcctaaaaaat -3 '；
B, extraction Nicotiana tabacum L. root tissue rna, reverse transcription obtains the first chain cdna；
C, basisntbrc1Gene order design synthesis specific primer, the first chain cdna being obtained using reverse transcription as template,
Carry out pcr amplification, from phusion high-fidelity amplification enzyme reaction system, system cumulative volume 50 μ l, comprising: 200ng cdna, 5
× phusion hf reaction buffer, the phusion high-fidelity dna polymerase of 10mm dntp, 2u,
Each 1 μm of forward and reverse primer, pcr reaction is carried out on mastercycler pro amplification instrument, and response procedures are: 98 DEG C, 30 seconds；
98 DEG C, 7 seconds, 55 DEG C, 30 seconds, 72 DEG C, 30 seconds, 35 circulations；72 DEG C extend 7 minutes；Reclaim and purification pcr product；
D, purified product are connected with carrier, are connected with topo carrier by test kit reaction, linked system is as follows with process: 4 μ l
Purified product+1 μ l salt salution+1 μ l pcr-blunt-topo mixes, 25 DEG C, water-bath 30min；By connected
Carrier is by heat-shock transformed entrance escherichia coli, plus is coated onto the lb containing 100mg/l kanamycin after fluid medium concussion and cultivate
Plate overnight is cultivated, and after growing bacterium, picking colony is cultivated, and takes 2ml bacterium solution to extract plasmid, carry out plasmid pcr after culture
Detection, screening positive clone, positive colony is sequenced.
Specific primers described in step c are:
Forward primer: 5 '-atgtatccgccaagcaacag -3 '；
Reverse primer: 5 '-gctattgaaatcctaaaaaat -3 '.
Culture medium described in Step d is: weighs tryptone 10g, yeast extract 5g, nacl 10g is dissolved in 1l
In distilled water, high temperature sterilize, that is, 121 DEG C, use after 25min sterilizing.
Tobacco axillary bud adjusting and controlling growth gene of the present inventionntbrc1Application be described tobacco axillary bud adjusting and controlling growth
Genentbrc1Application in obtaining the transfer-gen plant of the growth length that Nicotiana tabacum L. produces axillary bud after reduction is pinched.
Described acquisitionntbrc1The method of transgene tobacco comprises the following steps:
A, structure overexpression (oe) carrier:
A, with pcr-bluntii-topo carrier as intermediate carrier, pk2g7 is as expression vector establishmentntbrc1The table excessively of gene
Reach (oe) carrier, building primer is:
Ntbrc1-oe-f:5 '-ggatccatgtatccgccaagcaacag -3 ',
Ntbrc1- oe-r:5 '-ctcgagctattgaaatcctaaaaaat -3 ',
In ntbrc1- oe-f at ggatcc it isbamH i restriction enzyme site；At ctcgag in ntbrc1-oe-r it isxhoI enzyme action position
Point；
B, withntbrc1Positive colony topo plasmid dna carries out pcr amplification for template, and pcr reaction volume is 50 μ l, comprising:
200ng cdna, 5 × phusion hf reaction buffer, the phusion high-fidelity dna of 10mm dntp, 2u
Each 1 μm of polymerase, ntbrc1- oe-f primer, ntbrc1- oe-r primer, pcr reacts in mastercycler pro
Carry out on amplification instrument, response procedures are: 98 DEG C, 30 seconds；98 DEG C, 7 seconds, 55 DEG C, 30 seconds, 72 DEG C, 30 seconds, 30 circulations；72℃
Extend 7 minutes；Reclaim and purification pcr product；
C, recovery purpose fragment product are connected with topo carrier: reacted by test kit and be connected with topo carrier.Linked system with
Process is as follows: 4 μ l purified product+1 μ l salt salution+1 μ l pcr-blunt-topo mixes, 25 DEG C, water-bath
30min, by the carrier having connected by heat-shock transformed entrance escherichia coli, plus is coated onto card containing 100mg/l after culture medium concussion and cultivate
The lb plate overnight culture of that mycin, after growing bacterium, picking colony is cultivated, and takes 2ml bacterium solution to extract plasmid, enter after culture
Row plasmid pcr detects, screening positive clone is sequenced to positive colony；
D, entry clones pentrtm2b-ntbrc1The structure of oe: with topo plasmid and the pentr of pacing ordered pairtm2b empty carrier
Do endonuclease reaction:bamh i+xhoI double digestion topo plasmid and pentrtm2b-ccdb obtains genes of interest fragment and carrier segments
pentrtm2b, is attached after cutting glue reclaim, linked system: coupled reaction cumulative volume is 10 μ l, containing cutting of enzyme action purpose fragment
Glue reclaim product 4 μ l, pentrtm2b(bamhi+xhoI double digestion) carrier 1 μ l, 10 × t4 buffer 1 μ l, t4 ligase 1
μ l, sterilize distilled water 3 μ l, mixes, room temperature reaction 2 hours, is transformed into competent escherichia coli cell, plus culture medium concussion and cultivate
It is coated onto the lb plate overnight culture containing 100mg/l kanamycin afterwards, extract plasmid and carry out pcr detection and enzyme action detection, to test
Whether already inserted into carrier segments, whether entry vector successfully constructs card genes of interest fragment；
E, plant expression vector is obtained by lr reaction: select the correct entry clones of detection and plant expression vector pk2g7
(destination clone) carries out lr reaction, and concrete reaction is as follows: system is as follows with process:
1) connect entry clones (50-150ng) 1-7 μ l+0.5 μ l destination vector+te of 2b carrier
buffer(up to 8μl)；
2) above-mentioned system mixes, and ice bath 2min flicks 2 times；
3) 2 μ l lr clonease are addedtmEnzyme mix, to above-mentioned system, flicks, and mixes, centrifugation, 25 DEG C of water-baths
1h；
4) add 1 μ l proteinase k to flick, mix, 37 DEG C of water-bath 10min；
Proceed to escherichia coli by heat-shock transformed, after adding culture medium concussion and cultivate, apply the lb flat board containing 100mg/l spectinomycin
On, obtain plant expression vector, after choosing bacterium upgrading grain, carry out pcr checking and digestion verification.Pcr reaction volume is 25 μ l, bag
Include: 100ng plasmid dna, 5 × phusion hf reaction buffer, the phusion high-fidelity of 10mm dntp, 1u
Each 0.5 μm of dna polymerase, ntbrc1-oe-f primer, ntbrc1-oe-r primer, pcr reacts in mastercycler
Carry out on pro amplification instrument, response procedures are: 98 DEG C, 30 seconds；98 DEG C, 7 seconds, 55 DEG C, 30 seconds, 72 DEG C, 30 seconds, 30 circulations；72
DEG C extend 7 minutes；The correctness of detection recombiant plasmid, the clip size of gene upstream and downstream primer amplification is 987bp in theory, tests
Whether card pcr amplification is consistent with expection；Enzyme action system is 20 μ l: comprise plasmid dna 1 μ g,bamhi、xhoThe each 0.5 μ l of i,
Neb buffer 3.1 2 μ l, enzyme action testing result respectively carrier segments about 12kb and genes of interest fragment 999bp in theory,
Simultaneously meet two conditions, then show expression vector establishment success, by detection to plasmid carry out sequencing compare, test further
Card；
B, Agrobacterium-mediated Transformation:
Take out Agrobacterium competent cell from -80 DEG C of refrigerators, dissolve on ice, and add recombinant expression carrier pk7g-ntbrc1oe 4μl；Mixture is respectively put into liquid nitrogen flash freezer 1 minute, proceeds to 37 DEG C of water-baths 5 minutes, then ice bath 2 minutes, to mixed
In compound add 1ml lb fluid medium, 28 DEG C, 220rpm cultivate 3 ~ 4 hours；Culture is coated containing spectinomycin
On the lb solid medium of 100mg/l and rifampicin 25mg/l, it is inverted culture 2 ~ 3 days it is seen that containing the agriculture of purposeful carrier for 28 DEG C
Bacillus is cloned；
C, overexpression (oe) vector introduction Nicotiana tabacum L., culture transfer-gen plant:
A, picking contain the Agrobacterium colonies of destination carrier, in the flat lining out of the lb containing spectinomycin and rifampicin, 28 DEG C
Culture 2 ~ 3 days；Scraping line bacterial plaque connects bacterium in the ms culture medium containing spectinomycin and rifampicin, 28 DEG C, and 220rpm shakes
Culture, bacterial concentration reaches and is infected during od=0.5 ~ 0.8, obtains the Agrobacterium thalline containing destination carrier；Microorganism collection is arrived
In centrifuge tube, 6,000rpm centrifugation 5 minutes enrichment thalline, abandon supernatant, then with the resuspended thalline of 20ml liquid ms culture medium, contained
The Agrobacterium suspension bacteria liquid of destination carrier；
B, tobacco leaf is placed in 500ml wide mouthed bottle, adds appropriate 75% ethanol, rinse 1min；Outwell ethanol, add 0.1%
Hgcl2Solution, puts shaken at room temperature 15 ~ 30 minutes on shaking table；Outwell solution, with aseptic water washing 6 times；
C, tobacco leaf is taken out, wash away surface liquid with sterilizing absorbent paper, take aseptic blade shears to be cut into 1cm × 1cm's
Small pieces, the tobacco leaf cut into pieces is respectively put in the aseptic ms fluid medium suspension bacteria liquid containing destination carrier, standing
15~20min；Take out tobacco leaf, suck unnecessary bacterium solution with aseptic filter paper, in adding 6-ba 1.0mg/l, naa 0.1mg/l
25 DEG C of light culture two days in ms culture medium；Tobacco leaf is proceeded in division culture medium, incision contacts culture medium, in greenhouse experiment
Lower differentiation culture；Division culture medium is to add 6-ba 1.0mg/l, naa 0.1mg/l, kan 100mg/l, cephamycin
The ms culture medium of 500mg/l, is transferred in new culture medium, incision gradually grows calluss, finally divides for every 2 ~ 3 weeks
Dissolve bud；
D, the bud grown to 3 ~ 5cm is cut, proceed to ms culture medium root induction, the transfer-gen plant after taking root is by root media
Middle taking-up, with the net culture medium of tap water, transplants in the Nutrition Soil of sterilizing；
E, transfer-gen plant warpnptiiGene specific primer pcr checking amplification, identifies transgenic positive plant.
The hgcl of 0.1% described in step c b2Solution compound method is to weigh 0.1 gram of mercuric chloride, is dissolved with a little ethanol,
Add water and be settled to 100 milliliters.
To be embodied as case, the present invention will be further described below:
Embodiment 1ntbrc1The separating clone of gene
Using tomato dna sibrc1b(genbank accession number: hm597230) search tobacco gene group data base (https: //
Solgenomics.net/) obtain Nicotiana tabacum L.ntbrc1Nucleotide sequence.Entered according to this sequential design clone gene special primer
Row pcr reacts, and obtains purpose product；Purpose product is sequenced, obtainsntbrc1Gene order；Its nucleotides sequence is classified as seq id
Shown in no.1, encode 328 aminoacid, as shown in sequence table seqidno.2.
Extract cloud and mist 87 root rna, (invitrogen, by saying that this test kit provides using trizol test kit for extracting
Bright book operation).
2 μ g rna are taken to carry out reverse transcription, using primescript rt reagent kit with gdna eraser
(takara) test kit carries out genome dna removal reverse transcription (by the operation of test kit workbook).
The first chain cdna that reverse transcription is obtained, as template, expands for pcrntbrc1Full length gene, and design characteristicses
Row primer is as follows:
Forward primer: ntbrc1f:5 '-atgtatccgccaagcaacag -3 '；
Reverse primer: ntbrc1r:5 '-gctattgaaatcctaaaaaat -3 '.
Pcr reaction system is 50 μ l, comprising: 200ng cdna, 5 × phusion hf reaction buffer, 10mm dntp,
The phusion high-fidelity dna polymerase of 2u, each 1 μm of above-mentioned primer；Pcr reaction exists
Mastercycler pro(eppendorf, Germany) carry out on amplification instrument, response procedures are: 98 DEG C, 30 seconds；98 DEG C, 7 seconds,
55 DEG C, 30 seconds, 72 DEG C, 30 seconds, 35 circulations；72 DEG C extend 7 minutes.
Product is through 1%(g/ml) agarose gel electrophoresiies separate, amplification produce a single pcr band, see Fig. 1.
With qiaquick gel extraction kit(qiagen, Germany) reclaim amplified band, extraction step uses with reference to test kit
Explanation.The dna and topo carrier (invitrogen zero bluntii-topo-pcr cloning kit) of recovery purifying
Connect, by specification operates.Connection product utilizes heat shock method to convert escherichia coli, solid in the lb containing 100mg/l kanamycin
Screening positive clone in body flat board, 5 cloning and sequencings of picking (Dalian treasured biological engineering company limited).
Sequencing result showsntbrc1The total length of gene is 987bp, and its nucleotides sequence is classified as shown in seq id no:1, leads to
Cross sequencing, compare to determine it is the genes of interest that the present invention needs, this unnamed gene is by applicantntbrc1.ntbrc1Gene bag
Include the open reading frame of 987bp；328 aminoacid of coding.
Embodiment 2 overexpression (oe) vector construction
A, with pcr-bluntii-topo carrier as intermediate carrier, pk2g7 is as expression vector establishmentntbrc1The table excessively of gene
Reach (oe) carrier, building primer is:
Ntbrc1-oe-f:5 '-ggatccatgtatccgccaagcaacag -3 ',
Ntbrc1-oe-r:5 '-ctcgagctattgaaatcctaaaaaat -3 ',
At ggatcc in ntbrc1-oe-f it isbamH i restriction enzyme site；At ctcgag in ntbrc1-oe-r it isxhoI enzyme action position
Point；
B, withntbrc1Positive colony topo plasmid dna carries out pcr amplification for template, and pcr reaction volume is 50 μ l, comprising:
200ng cdna, 5 × phusion hf reaction buffer, the phusion high-fidelity dna of 10mm dntp, 2u
Each 1 μm of polymerase, ntbrc1-oe-f primer, ntbrc1-oe-r primer, pcr reacts in mastercycler pro
Carry out on amplification instrument, response procedures are: 98 DEG C, 30 seconds；98 DEG C, 7 seconds, 55 DEG C, 30 seconds, 72 DEG C, 30 seconds, 30 circulations；72℃
Extend 7 minutes；Reclaim and purification pcr product；
C, recovery purpose fragment product are connected with topo carrier: reacted by test kit and be connected with topo carrier.Linked system with
Process is as follows: 4 μ l purified product+1 μ l salt salution+1 μ l pcr-blunt-topo mixes, 25 DEG C, water-bath
30min.By the carrier having connected by thermal transition enter escherichia coli, plus culture medium concussion and cultivate after be coated onto card containing 100mg/l that
The lb plate overnight culture of mycin.After growing bacterium, picking colony is cultivated, and takes 2ml bacterium solution to extract plasmid, carry out after culture
Plasmid pcr detects.Screening positive clone, is sequenced to positive colony；
D, entry clones pentrtm2b-ntbrc1The structure of oe: with topo plasmid and the pentr of pacing ordered pairtm2b is unloaded
Body does endonuclease reaction:bamh i+xhoI double digestion topo plasmid and pentrtm2b-ccdb obtains genes of interest fragment and carrier-pellet
Section pentrtm2b, is attached after cutting glue reclaim, linked system: coupled reaction cumulative volume is 10 μ l, containing enzyme action purpose fragment
Cut glue reclaim product 4 μ l, pentrtm2b(bamh i+xhoI double digestion) carrier 1 μ l, 10 × t4 buffer 1 μ l, t4
Ligase 1 μ l, sterilize distilled water 3 μ l, mixes, room temperature reaction 2 hours, is transformed into competent escherichia coli cell, plus culture medium
It is coated onto the lb plate overnight culture containing 100mg/l kanamycin after concussion and cultivate, extract plasmid and carry out pcr detection and enzyme action
Detection, whether with verifying purpose genetic fragment already inserted into carrier segments, whether entry vector successfully constructs.
E, plant expression vector is obtained by lr reaction: select the correct entry clones of detection and plant expression vector
Pk2g7 (destination clone) carries out lr reaction, and concrete reaction is as follows: system is as follows with process:
1) connect entry clones (50-150ng) 1-7 μ l+0.5 μ l destination vector+te of 2b carrier
buffer(up to 8μl)
2) above-mentioned system mixes, and ice bath 2min flicks 2 times
3) 2 μ l lr clonease are addedtmEnzyme mix, to above-mentioned system, flicks, and mixes, centrifugation, 25 DEG C of water-baths
1h.
4) add 1 μ l proteinase k to flick, mix, 37 DEG C of water-bath 10min.
Proceed to escherichia coli by heat-shock transformed, after adding culture medium concussion and cultivate, apply the lb containing 100mg/l spectinomycin
On flat board.Obtain plant expression vector, after choosing bacterium upgrading grain, carry out pcr checking and digestion verification.Pcr reaction volume is 25 μ l,
Including: 100ng plasmid dna, 5 × phusion hf reaction buffer, the phusion high- of 10mm dntp, 1u
Fidelity dna polymerase, each 0.5 μm of ntbrc1-oe-f primer, ntbrc1-oe-r primer, pcr reaction exists
Carry out on mastercycler pro amplification instrument, response procedures are: 98 DEG C, 30 seconds；98 DEG C, 7 seconds, 55 DEG C, 30 seconds, 72 DEG C,
30 seconds, 30 circulations；72 DEG C extend 7 minutes；The correctness of detection recombiant plasmid, the piece of gene upstream and downstream primer amplification in theory
Duan great little is 987bp, and whether checking pcr amplification is consistent with expection；Enzyme action system is 20 μ l: comprise plasmid dna 1 μ g,bamh i、xhoThe each 0.5 μ l of i, neb buffer 3.1 2 μ l, enzyme action testing result respectively carrier segments about 12kb in theory
With genes of interest fragment 999bp.Simultaneously meet two conditions, then show expression vector establishment success, by detection to plasmid enter
Row sequencing compares, and verifies further.
The genetic transformation of embodiment 3 Nicotiana tabacum L.
(1) expression vector conversion Agrobacterium
Take out Agrobacterium competent cell from -80 DEG C of refrigerators, dissolve on ice, and add recombinant expression carrier pk2g7-ntbrc14μl；Mixture is respectively put into liquid nitrogen flash freezer 1 minute, proceeds to 37 DEG C of water-baths 5 minutes, then ice bath 2 minutes, to mixing
In thing add 1ml lb fluid medium, 28 DEG C, 220rpm cultivate 3 ~ 4 hours；Culture is coated containing spectinomycin
On the lb solid medium of 100mg/l and rifampicin 25mg/l, it is inverted culture 2 ~ 3 days it is seen that containing the agriculture of purposeful carrier for 28 DEG C
Bacillus is cloned；
(2) Transformation of tobacco
A, picking contain the Agrobacterium colonies of destination carrier, in the flat lining out of the lb containing spectinomycin and rifampicin, 28 DEG C
Culture 2 ~ 3 days；Scraping line bacterial plaque connects bacterium in the ms culture medium containing spectinomycin and rifampicin, 28 DEG C, and 220rpm shakes
Culture, bacterial concentration reaches and is infected during od=0.5 ~ 0.8, obtains the Agrobacterium thalline containing destination carrier；Microorganism collection is arrived
In centrifuge tube, 6,000rpm are enriched with thalline from 5 heart minutes, abandon supernatant, then with the resuspended thalline of 20ml liquid ms culture medium, are contained
The Agrobacterium suspension bacteria liquid of destination carrier；
B, tobacco leaf is placed in 500ml wide mouthed bottle, adds appropriate 75% ethanol, rinse 1min；Outwell ethanol, add 0.1%
Hgcl2Solution, puts shaken at room temperature 15 ~ 30 minutes on shaking table；Outwell solution, with aseptic water washing 6 times；
C, tobacco leaf is taken out, wash away surface liquid with sterilizing absorbent paper, take aseptic blade shears to be cut into 1cm × 1cm's
Small pieces, the tobacco leaf cut into pieces is respectively put in the aseptic ms fluid medium suspension bacteria liquid containing destination carrier, standing
15~20min；Take out tobacco leaf, suck unnecessary bacterium solution with aseptic filter paper, in adding 6-ba 1.0mg/l, naa 0.1mg/l
25 DEG C of light culture two days in ms culture medium；Tobacco leaf is proceeded in division culture medium, incision contacts culture medium, in greenhouse experiment
Lower differentiation culture；Division culture medium is to add 6-ba 1.0mg/l, naa 0.1mg/l, kan 100mg/l, cephamycin
The ms culture medium of 500mg/l, is transferred in new culture medium, incision gradually grows calluss, finally divides for every 2 ~ 3 weeks
Dissolve bud；
D, the bud grown to 3 ~ 5cm is cut, proceed to ms culture medium root induction, the transfer-gen plant after taking root is by root media
Middle taking-up, with the net culture medium of tap water, transplants in the Nutrition Soil of sterilizing；
E, transfer-gen plant warpnptiiGene specific primer pcr checking amplification, identifies transgenic positive plant.
Embodiment 4 transfer-gen plant is pinched test
After transfer-gen plant is transplanted to disposable flowerpot, after growing 16 days, pinch, measure the length of each axillary bud, result is shown in Fig. 6.
Overexpression (oe) plant reduces about 60-75% than empty vector control vector control (vc).The above results show,ntbrc1
Gene is responsible for the genetic manipulation controlling the length of tobacco axillary bud growth to pass through to this gene, can reduce the growth of tobacco axillary bud,
The exploitation of this gene advantageously reduce the use of Suckering agents in leaf tobacco production.
sequence listing
<110>Yunnan Academy of Tobacco Agricultural Science
<120>a kind of tobacco axillary bud adjusting and controlling growth gene ntbrc1 and its cloning process and application
<130> 2016
<160> 6
<170> patentin version 3.3
<210> 1
<211> 987
<212> dna
<213>nucleotide sequence of gene ntbrc1
<400> 1
atgtatccgc caagcaacag ctgcaactac agccccattt tcaacatccc ttctccttgt 60
atgcaatatg gagacgaact attcttccaa tattatcctg accatttcct tcaacagcaa 120
caagtgcctt tgatagaaga tcagagtgtt gacatcttag ctgattgcac tgagaatgtt 180
actaacgaag aaactgtcat caatactgat actgtaaaag ttctttatga cacaggagct 240
gttacaaaca gtcagtgttg gggaggaaat gaagaagtag aagaaggccg cgaaaacaaa 300
agaaatgaca tgagaagcac cattagtatt attcatgtac ggaaaaacaa gaaatgttcc 360
aataaagatc gacatagcaa gattaacact gctcgtggcc tcagagaccg aaggatgaga 420
ctttcccttg atgcagctcg caagtttttc agtttacaag acatgttggg gtccgataag 480
gcaagtaaaa ctgtagaatg gttgcttatc aaatcggggt ctgaaatcga agagctagcc 540
aaaggcaata aaggaggagg cattcctaaa caaagctgca gtactactaa tggaattggt 600
gcaattagta ctgcaatatc ctctatttct gagtgtgagg ttatatcagg aactgatgaa 660
tctttctcta ttacttataa aaagaagctg aaaactgcta aaggagcctc gaaaaagacg 720
gctaaaactg ctcgtagagc tgcatttgat cgtcttatta caagggaaac gaggaatcaa 780
gcaagggcta gggctagaga gagaacaaaa ataaagaaaa gcctcggtaa atccaaagag 840
aacagtgctg attactgtaa tttggtggat aattatggag attggagtca atttagtatc 900
ttcaactatc agaaaaatgc agttggaatt tcccatgatc aggtgggttc aataattaaa 960
caacatgatt ttttaggatt tcaatag 987
<210> 2
<211> 328
<212> prt
<213>aminoacid sequence of gene ntbrc1 coding
<400> 2
met tyr pro pro ser asn ser cys asn tyr ser pro ile phe asn ile
1 5 10 15
pro ser pro cys met gln tyr gly asp glu leu phe phe gln tyr tyr
20 25 30
pro asp his phe leu gln gln gln gln val pro leu ile glu asp gln
35 40 45
ser val asp ile leu ala asp cys thr glu asn val thr asn glu glu
50 55 60
thr val ile asn thr asp thr val lys val leu tyr asp thr gly ala
65 70 75 80
val thr asn ser gln cys trp gly gly asn glu glu val glu glu gly
85 90 95
arg glu asn lys arg asn asp met arg ser thr ile ser ile ile his
100 105 110
val arg lys asn lys lys cys ser asn lys asp arg his ser lys ile
115 120 125
asn thr ala arg gly leu arg asp arg arg met arg leu ser leu asp
130 135 140
ala ala arg lys phe phe ser leu gln asp met leu gly ser asp lys
145 150 155 160
ala ser lys thr val glu trp leu leu ile lys ser gly ser glu ile
165 170 175
glu glu leu ala lys gly asn lys gly gly gly ile pro lys gln ser
180 185 190
cys ser thr thr asn gly ile gly ala ile ser thr ala ile ser ser
195 200 205
ile ser glu cys glu val ile ser gly thr asp glu ser phe ser ile
210 215 220
thr tyr lys lys lys leu lys thr ala lys gly ala ser lys lys thr
225 230 235 240
ala lys thr ala arg arg ala ala phe asp arg leu ile thr arg glu
245 250 255
thr arg asn gln ala arg ala arg ala arg glu arg thr lys ile lys
260 265 270
lys ser leu gly lys ser lys glu asn ser ala asp tyr cys asn leu
275 280 285
val asp asn tyr gly asp trp ser gln phe ser ile phe asn tyr gln
290 295 300
lys asn ala val gly ile ser his asp gln val gly ser ile ile lys
305 310 315 320
gln his asp phe leu gly phe gln
325
<210> 3
<211> 20
<212> dna
<213>forward primer
<400> 3
atgtatccgc caagcaacag 20
<210> 4
<211> 21
<212> dna
<213>reverse primer
<400> 4
gctattgaaa tcctaaaaaa t 21
<210> 5
<211> 26
<212> dna
<213> ntbrc1-oe-f
<400> 5
ggatccatgt atccgccaag caacag 26
<210> 6
<211> 26
<212> dna
<213> ntbrc1- oe-r
<400> 6
ctcgagctat tgaaatccta aaaaat 26
Claims (8)
1. a kind of tobacco axillary bud adjusting and controlling growth gene ntbrc1 is it is characterised in that described tobacco axillary bud adjusting and controlling growth gene
The nucleotide sequence of ntbrc1 is as shown in seq id no:1.
2. tobacco axillary bud adjusting and controlling growth gene ntbrc1 according to claim 1 is it is characterised in that described tobacco axillary bud
The aminoacid sequence of adjusting and controlling growth gene ntbrc1 coding is as shown in seq id no:2.
3. a kind of cloning process of the tobacco axillary bud adjusting and controlling growth gene ntbrc1 described in claim 1 is it is characterised in that include
Following steps:
A, determinationntbrc1Gene order；
(1) obtain Nicotiana tabacum L. using tomato dna sibrc1b search tobacco gene group data basentbrc1Nucleotide sequence, utilize
This sequential design gene cloning primer:
Forward primer: 5 '-atgtatccgccaagcaacag -3 '；
Reverse primer: 5 '-gctattgaaatcctaaaaaat -3 '；
B, extraction Nicotiana tabacum L. root tissue rna, reverse transcription obtains the first chain cdna；
C, basisntbrc1Gene order design synthesis specific primer, the first chain cdna being obtained using reverse transcription as template,
Carry out pcr amplification, from phusion high-fidelity amplification enzyme reaction system, system cumulative volume 50 μ l, comprising: 200ng cdna, 5
× phusion hf reaction buffer, the phusion high-fidelity dna polymerase of 10mm dntp, 2u,
Each 1 μm of forward and reverse primer, pcr reaction is carried out on mastercycler pro amplification instrument, and response procedures are: 98 DEG C, 30 seconds；
98 DEG C, 7 seconds, 55 DEG C, 30 seconds, 72 DEG C, 30 seconds, 35 circulations；72 DEG C extend 7 minutes；Reclaim and purification pcr product；
D, purified product are connected with carrier, are connected with topo carrier by test kit reaction, linked system is as follows with process: 4 μ l
Purified product+1 μ l salt salution+1 μ l pcr-blunt-topo mixes, 25 DEG C, water-bath 30min；By connected
Carrier is by heat-shock transformed entrance escherichia coli, plus is coated onto the lb containing 100mg/l kanamycin after fluid medium concussion and cultivate
Plate overnight is cultivated, and after growing bacterium, picking colony is cultivated, and takes 2ml bacterium solution to extract plasmid, carry out plasmid pcr after culture
Detection, screening positive clone, positive colony is sequenced.
4. the cloning process of tobacco axillary bud adjusting and controlling growth gene ntbrc1 according to claim 3 is it is characterised in that step c
Described in specific primers be:
Forward primer: 5 '-atgtatccgccaagcaacag -3 '；
Reverse primer: 5 '-gctattgaaatcctaaaaaat -3 '.
5. tobacco axillary bud adjusting and controlling growth gene according to claim 3ntbrc1Cloning process it is characterised in that Step d
Described in culture medium be: weigh tryptone 10g, yeast extract 5g, nacl 10g is dissolved in 1l distilled water, high temperature
Sterilizing, uses after 25min sterilizing by that is, 121 DEG C.
6. the tobacco axillary bud adjusting and controlling growth gene described in a kind of claim 1ntbrc1Application it is characterised in that described cigarette
Careless axillary bud growth controlling genentbrc1In obtaining the transfer-gen plant of the growth length that Nicotiana tabacum L. produces axillary bud after reduction is pinched
Application.
7. tobacco axillary bud adjusting and controlling growth gene according to claim 6ntbrc1Application it is characterised in that described acquisitionntbrc1The method of transgene tobacco comprises the following steps:
A, structure overexpression (oe) carrier:
A, with pcr-bluntii-topo carrier as intermediate carrier, pk2g7 is as expression vector establishmentntbrc1The table excessively of gene
Reach (oe) carrier, building primer is:
Ntbrc1-oe-f:5 '-ggatccatgtatccgccaagcaacag -3 ',
Ntbrc1- oe-r:5 '-ctcgagctattgaaatcctaaaaaat -3 ',
In ntbrc1- oe-f at ggatcc it isbamH i restriction enzyme site；At ctcgag in ntbrc1-oe-r it isxhoI enzyme action position
Point；
B, withntbrc1Positive colony topo plasmid dna carries out pcr amplification for template, and pcr reaction volume is 50 μ l, comprising:
200ng cdna, 5 × phusion hf reaction buffer, the phusion high-fidelity dna of 10mm dntp, 2u
Each 1 μm of polymerase, ntbrc1- oe-f primer, ntbrc1- oe-r primer, pcr reacts in mastercycler pro
Carry out on amplification instrument, response procedures are: 98 DEG C, 30 seconds；98 DEG C, 7 seconds, 55 DEG C, 30 seconds, 72 DEG C, 30 seconds, 30 circulations；72℃
Extend 7 minutes；Reclaim and purification pcr product；
C, recovery purpose fragment product are connected with topo carrier: reacted by test kit and be connected with topo carrier；
Linked system is as follows with process: 4 μ l purified product+1 μ l salt salution+1 μ l pcr-blunt-topo
Mix, 25 DEG C, water-bath 30min, by the carrier having connected by heat-shock transformed entrance escherichia coli, plus apply after culture medium concussion and cultivate
To the lb plate overnight culture containing 100mg/l kanamycin, after growing bacterium, picking colony is cultivated, and takes 2ml after culture
Bacterium solution extracts plasmid, carries out plasmid pcr detection, screening positive clone, positive colony is sequenced；
D, entry clones pentrtm2b-ntbrc1The structure of oe: with topo plasmid and the pentr of pacing ordered pairtm2b empty carrier
Do endonuclease reaction:bamh i+xhoI double digestion topo plasmid and pentrtm2b-ccdb obtains genes of interest fragment and carrier-pellet
Section pentrtm2b, is attached after cutting glue reclaim, linked system: coupled reaction cumulative volume is 10 μ l, containing enzyme action purpose fragment
Cut glue reclaim product 4 μ l, pentrtm2b(bamhi+xhoI double digestion) carrier 1 μ l, 10 × t4 buffer 1 μ l, t4
Ligase 1 μ l, sterilize distilled water 3 μ l, mixes, room temperature reaction 2 hours, is transformed into competent escherichia coli cell, plus culture medium
It is coated onto the lb plate overnight culture containing 100mg/l kanamycin after concussion and cultivate, extract plasmid and carry out pcr detection and enzyme action
Detection, whether with verifying purpose genetic fragment already inserted into carrier segments, whether entry vector successfully constructs；
E, plant expression vector is obtained by lr reaction: select the correct entry clones of detection and plant expression vector pk2g7
(destination clone) carries out lr reaction, and concrete reaction is as follows: system is as follows with process:
1) connect entry clones (50-150ng) 1-7 μ l+0.5 μ l destination vector+te of 2b carrier
buffer(up to 8μl)；
2) above-mentioned system mixes, and ice bath 2min flicks 2 times；
3) 2 μ l lr clonease are addedtmEnzyme mix, to above-mentioned system, flicks, and mixes, centrifugation, 25 DEG C of water-bath 1h；
4) add 1 μ l proteinase k to flick, mix, 37 DEG C of water-bath 10min；
Proceed to escherichia coli by heat-shock transformed, after adding culture medium concussion and cultivate, apply the lb flat board containing 100mg/l spectinomycin
On, obtain plant expression vector, after choosing bacterium upgrading grain, carry out pcr checking and digestion verification；
Pcr reaction volume is 25 μ l, comprising: 100ng plasmid dna, 5 × phusion hf reaction buffer, 10mm dntp, 1u
Phusion high-fidelity dna polymerase, ntbrc1-oe-f primer, ntbrc1-oe-r primer each 0.5
μm, pcr reaction is carried out on mastercycler pro amplification instrument, and response procedures are: 98 DEG C, 30 seconds；98 DEG C, 7 seconds, 55
DEG C, 30 seconds, 72 DEG C, 30 seconds, 30 circulations；72 DEG C extend 7 minutes；The correctness of detection recombiant plasmid, gene is upper and lower in theory
The clip size of trip primer amplification is 987bp, and whether checking pcr amplification is consistent with expection；Enzyme action system is 20 μ l: comprises
Plasmid dna 1 μ g,bamhi、xhoThe each 0.5 μ l of i, neb buffer 3.1 2 μ l, in theory enzyme action testing result be respectively carrier
Fragment about 12kb and genes of interest fragment 999bp, meet two conditions simultaneously, then show expression vector establishment success, will detect
To plasmid carry out sequencing compare, verify further；
B, Agrobacterium-mediated Transformation:
Take out Agrobacterium competent cell from -80 DEG C of refrigerators, dissolve on ice, and add recombinant expression carrier pk7g-ntbrc1oe 4μl；Mixture is respectively put into liquid nitrogen flash freezer 1 minute, proceeds to 37 DEG C of water-baths 5 minutes, then ice bath 2 minutes, to mixed
In compound add 1ml lb fluid medium, 28 DEG C, 220rpm cultivate 3 ~ 4 hours；Culture is coated containing spectinomycin
On the lb solid medium of 100mg/l and rifampicin 25mg/l, it is inverted culture 2 ~ 3 days it is seen that containing the agriculture of purposeful carrier for 28 DEG C
Bacillus is cloned；
C, overexpression (oe) vector introduction Nicotiana tabacum L., culture transfer-gen plant:
A, picking contain the Agrobacterium colonies of destination carrier, in the flat lining out of the lb containing spectinomycin and rifampicin, 28 DEG C
Culture 2 ~ 3 days；Scraping line bacterial plaque connects bacterium in the ms culture medium containing spectinomycin and rifampicin, 28 DEG C, and 220rpm shakes
Culture, bacterial concentration reaches and is infected during od=0.5 ~ 0.8, obtains the Agrobacterium thalline containing destination carrier；Microorganism collection is arrived
In centrifuge tube, 6,000rpm centrifugation 5 minutes enrichment thalline, abandon supernatant, then with the resuspended thalline of 20ml liquid ms culture medium, contained
The Agrobacterium suspension bacteria liquid of destination carrier；
B, tobacco leaf is placed in 500ml wide mouthed bottle, adds appropriate 75% ethanol, rinse 1min；Outwell ethanol, add 0.1%
Hgcl2Solution, puts shaken at room temperature 15 ~ 30 minutes on shaking table；Outwell solution, with aseptic water washing 6 times；
C, tobacco leaf is taken out, wash away surface liquid with sterilizing absorbent paper, take aseptic blade shears to be cut into 1cm × 1cm's
Small pieces, the tobacco leaf cut into pieces is respectively put in the aseptic ms fluid medium suspension bacteria liquid containing destination carrier, standing
15~20min；Take out tobacco leaf, suck unnecessary bacterium solution with aseptic filter paper, in adding 6-ba 1.0mg/l, naa 0.1mg/l
25 DEG C of light culture two days in ms culture medium；Tobacco leaf is proceeded in division culture medium, incision contacts culture medium, in greenhouse experiment
Lower differentiation culture；Division culture medium is to add 6-ba 1.0mg/l, naa 0.1mg/l, kan 100mg/l, cephamycin
The ms culture medium of 500mg/l, is transferred in new culture medium, incision gradually grows calluss, finally divides for every 2 ~ 3 weeks
Dissolve bud；
D, the bud grown to 3 ~ 5cm is cut, proceed to ms culture medium root induction, the transfer-gen plant after taking root is by root media
Middle taking-up, with the net culture medium of tap water, transplants in the Nutrition Soil of sterilizing；
E, transfer-gen plant warpnptiiGene specific primer pcr checking amplification, identifies transgenic positive plant.
8. tobacco axillary bud adjusting and controlling growth gene according to claim 7ntbrc1Application it is characterised in that in step c b
The hgcl of described 0.1%2Solution compound method is to weigh 0.1 gram of mercuric chloride, with the dissolving of a little ethanol, adds water and is settled to 100 millis
Rise.
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| 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 |
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| WO2016057515A2 (en) * | 2014-10-06 | 2016-04-14 | Altria Client Services Llc | Genetic control of axillary bud growth in tobacco plants |
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| WO2016057515A2 (en) * | 2014-10-06 | 2016-04-14 | Altria Client Services Llc | Genetic control of axillary bud growth in tobacco plants |
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| 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 |
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