CN108192896A - The slow anion channel albumen NtSLAH1 of one tobacco and its application - Google Patents
The slow anion channel albumen NtSLAH1 of one tobacco and its application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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- C07K—PEPTIDES
- 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)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—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
- 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
Abstract
The invention belongs to transgenic tobacco technical fields, and in particular to the slow anion channel albumen NtSLAH1 of a tobacco and its using patent application.Gene C DS sequences include 1107bp bases, and base sequence is as shown in SEQ ID NO.1；Wherein the 270th 615 nucleotide are nucleic acid specific fragment.The slow anion channel albumen NtSLAH1 of tobacco is the key protein of tobacco chlorion metabolism, passes through virus induced gene silencing（VIGS）Technology, inhibitNtSLAH1After gene expression, chloride ion content is substantially reduced in transgene silencing plant.Can be to cultivate low chlorine content new product of tobacco to establish good basis, while also provide the technical support of essence for the stabilization of quality of tobacco and cigarette quality improvement based on this.
Description
Technical field
The invention belongs to transgenic tobacco technical fields, and in particular to the slow anion channel albumen of a tobacco
NtSLAH1 and its using patent application.
Background technology
Existing research for tobacco reaches in content it is believed that the chloride ion content in tobacco leaf is advisable with 0.3 ~ 0.8
It can influence to glow when 1% to hold fire, further above 1% when will appear grey black and stop working phenomenon；On the other hand, chlorion contains in tobacco leaf
Starch accumulation can be caused more when measuring excessively high, blade is plump and crisp, and hygroscopicity is big so that color easily deepens during storage, generates bad
Smell.In short, chloride ion content has more important directly affect for quality of tobacco in tobacco leaf.
Based on chloride ion content in tobacco leaf to cigarette quality directly affect and its importance, some investigators are to the whole nation
Chloride ion content has carried out detection statistics in the tobacco leaf of various regions, the results showed that（Zhengzhou Tobacco Research Institute of CNTC,《In
State's quality of tobacco white paper（2015）》）：Chloride ion content in 2011 ~ 2015 cured tobacco leaf chemical compositions in Henan
（0.53%-0.65%）Far above national mean value（0.26%-0.30%）.In addition, according to Shang Yan groups to cigarette district 2012-2015 in Henan
The average value of year chloride content determination, especially 2014 annual datas（Inferior leads 2.21%, middle leaf 2.09% and upper leaf
2.03%）, all significantly larger than the whole nation is horizontal for chloride ion content in recent years for cigarette district in Henan.These statistical data show some areas cigarette
The inhomogeneity of chloride ion content even higher feature in leaf quality inhomogeneity, especially tobacco leaf, it has also become restrict Luzhou-flavor
One of bottleneck that quality of tobacco improves.
To solve the problems, such as that tobacco leaf chloride ion content is higher, traditional improvement mode is mostly started with from cultivation technique, by excellent
Change field management measure, improve modulation fermentation technique to stablize and promote quality of tobacco.But in general, these measures not from
Fundamentally change the situation that these qualities of tobacco are relatively low, industrial applicibility is not strong.So that related corrective measure lacks preferably
Practicability.
With the fast development of genomics especially gene editing technology so that people are for chloride ions accumulated in tobacco leaf
Relationship understanding between related gene is more and more deep.Based on it is existing research it is known that be：Slow anion channel
（SLAC）Protein family is mainly in plant anion（Chlorion and nitrate ion etc.）It is played during intake and stomatal movement
Important function.But due toSLACGene family gene is more, functions and differs greatly in different plants, thus for difference
Plant, differenceSLACThe function of gene is it is still necessary to conduct further research and distinguish, so as to targetedly in the future
Plant improvement.
Invention content
Present invention aims at provide the slow anion channel albumen NtSLAH1 of a tobacco（Slowly activating
Anion Channel Homologue, SLAH）, the slow anion channel albumen NtSLAH1 and Cl of tobacco of the coded by said gene-
Absorption and transport it is related, can be that the new product of tobacco of low chlorine ion content lay the foundation based on this function.
Details are as follows for the technical solution that the application is taken.
The encoding gene of the slow anion channel albumen NtSLAH1 of tobaccoNtSLAH1Gene, the gene source is in tobacco
（Nicotiana tabacum）, CDS sequences include 1107bp bases, base sequence is as shown in SEQ ID NO.1；Wherein
270th -615 nucleotide are nucleic acid specific fragment.
Described in PCR amplification obtainsNtSLAH1The method of gene specifically refers to as follows：
（1）The total serum IgE of tobacco sample is extracted, and reverse transcription is spare for cDNA；
（2）It is as follows to design amplimer sequence：
F：5 '-CGCGAGCTCGGTACCATGGGGGAAGAAGTTTTTG-3 ',
R：5’-GCTCACCATGGATCCCTAATTACGTTTAGTGAAGT-3’；
With step（1）In prepared cDNA be template, utilize above-mentioned primer carry out PCR amplification.
The slow anion channel albumen NtSLAH1 albumen of tobacco, is a kind of ionophorous protein, which transports with chlorion
Correlation, including 368 amino acid, amino acid sequence is as shown in SEQ ID NO.2.
Applications of the slow anion channel albumen NtSLAH1 of tobacco in tobacco, for transporting chlorion.
The encoding gene of the slow anion channel albumen NtSLAH1 of tobaccoNtSLAH1Application of the gene in tobacco, will
After the gene silencing, chloride ion content is decreased obviously in plant.
For the encoding gene of the slow anion channel albumen NtSLAH1 of silence tobaccoNtSLAH1The instantaneous silence of gene
VIGS carriers, construction method are：Utilize virus induced gene silencing（VIGS）Technology, with the slow anion channel of the tobacco
The encoding gene of albumen NtSLAH1NtSLAH1The specific nucleotide acid fragment of gene is as boot sequence, by the specific nucleic acid
Segment is connected on transient expression vector TRV, and after converting bacillus coli DH 5 alpha, further through screening, identifying, structure obtains instantaneous
Silence VIGS carriers：TRV-NtSLAH1。
The encoding gene for the slow anion channel albumen NtSLAH1 of silence tobaccoNtSLAH1The instantaneous of gene is sunk
Silent application of the VIGS carriers in tobacco, using transgenic technology, after the VIGS carrier transformation of tobacco plant, for silenceNtSLAH1Gene so that the slow anion channel albumen NtSLAH1 protein gene expression amounts of tobacco are substantially reduced or even can not table
It reaches, it is final to reduce chloride ion content in plant.
A kind of method for cultivating low chlorine plant variety using transgenic technology, described will be used for the slow anion of silence tobacco
The encoding gene of channel protein NtSLAH1NtSLAH1The instantaneous silence VIGS carriers conversion plant of gene, screening, identification obtain
Obtain instantaneous silence plant；Further, the method interfered using RNAi builds gene silencing vector, converts Nicotiana tabacum, passes through
Screening, identification can obtain genetically modified plants new varieties, in genetically modified plants new varietiesNtSLAH1The expression of gene is limited
System；The plant to be transformed is common cultivation tobacco.
The slow anion channel albumen NtSLAH1 of tobacco in the present invention is the key protein of tobacco chlorion metabolism, is passed through
Real-time PCR have found shouldNtSLAH1Gene is all expressed in each tissue of tobacco, and in the expression of tobacco root relatively
It is high.To further confirm that the protein function, pass through virus induced gene silencing（VIGS）Technology, construct silenceNtSLAH1The VIGS carriers of gene, have successfully been obtained inhibition after conversionNtSLAH1The transgenosis expressed in Ben's tobacco is sunk
Silent plant.Testing result shows relative comparison plant, and chloride ion content is substantially reduced in transgene silencing plant, reduce to
Few 35%, i.e.,：The transgene silencing plant obtained possesses the specific phenotypes that chloride ion content relative comparison plant is substantially reduced,
In other words, silenceNtSLAH1The chloride ion content in plant can be significantly reduced after gene.
It can be seen that with reference to existing technique for gene engineering and pass through knockout or silence using gene silent technologyNtSLAH1
After gene, the chloride ion content that can be substantially reduced in plant can be to cultivate low chlorine content new product of tobacco establish based on this
Good basis, while the technical support of essence is also provided for the stabilization of quality of tobacco and cigarette quality improvement.
Description of the drawings
Fig. 1 is in different tissues organNtSLAH1Relative expression quantity；
Fig. 2 is in silence plantNtSLAH1 The relative expression quantity of gene；
Fig. 3 is the chloride ion content in each processing group tobacco leaf after drying.
Specific embodiment
Explanation is further explained to the application with reference to embodiment, before specific embodiment is introduced, with regard to following realities
The basic conditions such as involved part biological material, experiment reagent, laboratory apparatus in example are applied to be briefly discussed below.
Biomaterial：
Tobacco-containing material：Ben's tobacco（Nicotiana benthamiana）, a kind of commercialization tobacco bred；
Interference carrier：TRV, purchased from Chinese plasmid vector bacterium cytogene collection；
Gene sequencing and primer synthesis provide completion by Shanghai life work；
Experiment reagent：
LA Taq enzymes, PstI restriction enzymes, plasmid extraction kit, plastic recovery kit etc., purchased from Takara companies,
In-Fusion T-A clone kits, purchased from clontech companies；
RNA extracts kits, purchased from GeneAnswer companies；
Reverse transcription reagent box, RT-PCR kit, purchased from Roche companies；
Peptone, yeast extract etc., purchased from Oxoid companies；
Portion of reagent formula and preparation method are briefly described as follows：
（1）LB fluid nutrient mediums（1L）：10 g bacto peptones（bacteriological peptone）, 10 g sodium chloride
（NaCl）, 5 g yeast extracts（yeast extract）, autoclave sterilization；
（2）YEB fluid nutrient mediums（1L）：5 g beef extracts（beef extract）, 5 g bacto peptones
（bacteriological peptone）, 5 g sucrose（sucrose）, 1 g yeast extracts（yeast extract）, 2
Ml 1M magnesium sulfate（MgSO4）, autoclave sterilization；
（3）1M 2- (N- morpholines) ethanesulfonic acid（MES）Storing solution：ddH2O dissolves MES, and filtration sterilization, -20 DEG C store for future use；
（4）200 mM acetosyringones（Acetosyringone）Storing solution：Dimethyl sulfoxide (DMSO)（DSMO）Dissolve acetyl cloves
Ketone, -20 DEG C store for future use；
（5）MMA（1L）：20 g sucrose（sucrose）, 5 g MS salts（Duchefa Biochemie）, 1.95 g
MES, 1 ml acetosyringones（200 mM,）, pH=5.6；
Laboratory apparatus：
PCR instrument Tgradient, Biometra Products,
Real-time PCR LightCycler 96, Roche Products.
Embodiment 1
The present embodiment is mainly with regard to the slow anion channel albumen of tobaccoNtSLAH1The acquisition process of gene, is briefly discussed below.
Using cultigen tobacco leaf as sample, tobacco leaf total serum IgE is extracted using RNA extracts kits, reverse transcription is
CDNA is spare；
By the method for homologous comparison, with reference to arabidopsisAtSLAH1The sequence of gene and known tobacco section gene order, design
Amplimer sequence is as follows：
F：5 '-CGCGAGCTCGGTACCATGGGGGAAGAAGTTTTTG-3 ',
R：5’-GCTCACCATGGATCCCTAATTACGTTTAGTGAAGT-3’；
Using above-mentioned prepared cDNA as template, PCR amplification is carried out using above-mentioned primer.
During PCR amplification, 50 μ L reaction system Reference Designs are as follows：
Sense primer F, 1 μ L；
Downstream primer R, 1 μ L；
CDNA templates, 1 μ L；
10 × buffer, 5 μ L；
DNTP, 6 μ L；
EazyTaq enzymes, 1 μ L；
Add ddH2O to 50 μ L；
PCR response procedures：94 DEG C of pre-degeneration 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 45s, 72 DEG C of extension 1min, 35 recycle；
72 DEG C of extension 10min.
PCR product recycles purpose band after the detection of 1% agarose gel electrophoresis.Further, using In-Fusion side
PCR product is connect, and convert E. coli competent DH5 α with plant expression vector pFF19 after method will purify, and 37 DEG C were cultivated
Night.After screening and identification, it is sequenced to converting after correct plasmid purifies, obtains the slow anion channel albumen of tobacco
The encoding gene of NtSLAH1NtSLAH1Gene, altogether including 1107bp base, analysis shows wherein the 270th -615 nucleosides
Acid is nucleic acid specific fragment, and base sequence is specific as follows as shown in SEQ ID NO.1：
ATGGGGGAAGAAGTTTTTGAATCAACAATCAAAGTCACAATAAGTGATGATACTATGAAATCAAATGTTGCCA
AGAAATCATCTTCTGCTTCTCTTTTGACTAAACTACATGCAGGCTATTTCAGAATAAGCCTATCTTTAGGTGGTCAA
GCCTTGTTATGGAAAGTTCTAATTGAACATTTAGATAAATCACAAACTCTTCACCACTTATTTCATACTCTCCCTTC
AACTACTTTCCTCTTACTATGGTGGATTTCCCTTTGTACCCTTCTCATCCTCTCTTTTCTTTACATTTTAAGGTGCA
TTTTTCACTTCTCATTAGTTAAATCAGAGTTTTTACATCCTGTTGGTGTAAACTATCTCTTTGCTCCTTGGATTTCT
TGGCTTTTATTACTCCAATCAGCACCATTTAGTATTCCACATGTTGGTTCTTGCCAAGTTTTTTGGTGGATTTTCGT
CGTTCCGGTTGGGATTCTTGATGTGAAAATATATGGACAATGGTTTACTACTGAGAAAAGGTTTTTATCAATGGTTG
CAAATCCAACTAGCCAACTTTCTGTGTTGGGAAATTTGACTGGTGCTTGGGTTGCAAGTAAAAAGGAATGGAAAGAG
AGTGCTGTTTGTATATTTACATTAGGGTTAACACATTATTTGGTAGTGTTTATTACACTTTATCAAAGATTATCTGG
TAGTAATAACCTACCTGCTATGCTTAGACCTTCTTTCTTTTTGTTTGTGGCTGCTCCTAGTATGGCTAGCTTAGCTT
GGGCTTCTATTTCTGGGAATTTTGATATGTCATGCAGAATGCTCTTTTTTCTCTCACTATTTCTCTTCACTTCTTTG
GTTTGTAGGCCAGCACTATTCAAGAAATCAATGAGAAAGTTCAATGTTGCATGGTGGGCTTACTCATTTCCTCTCAC
ATTCCTAGCCTTAGCCTCTGCACAATATGCACATCAAGTGAAAGGTCCTGTTTCTGCTGGACTTATGCTGCTTCTCT
CAGCCCTTTCAGTTCTTGTTTTTGTTGGTTTGACAGTTTCCACTGCTCTCAATCTTGACATGCTTTTGGCTGATAAT
GATCGCTATTTAAACTTCACTAAACGTAATTAG。
To encoding geneNtSLAH1Gene analyzed, translate after, it is known that the slow anion channel albumen NtSLAH1 of tobacco
Amino acid sequence, which includes 368 amino acid altogether, and amino acid sequence is specific as follows as shown in SEQ ID NO.2：
MGEEVFESTIKVTISDDTMKLDVAKKPSSASLLTKLHAGYFRISLSLGGQALLWKVLIEHLDKSKTLHHLFHT
LPSTTFLLLWWISLCTLIILSFLYILRCIFHFSLVKSEFLHPVGVNYLFAPWISWLLLLQSAPFSIPHVGSCQVFWW
IFVIPVGILDVKIYGQWFTTEKRFLSMVANPTSQLSVLGNLTGAWIASKKEWKESAVCIFTLGLTHYLVVFITLYQR
LSGSNSLPAMLRPSFFLFVAAPSMASLAWASISGDFDMSCRMLFFLSLFLFTSLVCRPAIFKKSMKKFNVAWWSYSF
PLTFLALASVQYAHQVKSPVSAGLMLLLSALSVLVFVGLTVSTALNLDMLLADNDRYLNFTKRN。
In order to further appreciate thatNtSLAH1Expression specificity of the gene in different tissues, utilizes real-time PCR
Technology, respectively to cultivate the organs such as cigarette seed, maturity period tobacco leaf, stem, root, leaf bud, stamen, gynoecium and calyx as sample,
It is rightNtSLAH1Expression of the gene in different tissues is detected.The results are shown in Figure 1.
Analysis can be seen thatNtSLAH1Gene has expression, but the table in tobacco root in each tissue of tobacco
Up to amount highest.
Embodiment 2
It is determiningNtSLAH1Function of the gene in tobacco, selectionNtSLAH1Specific nucleic acid segment in gene（Sequence table SEQ
The 270th -615 nucleotide sequences of ID NO.1）As boot sequence, silence is constructedNtSLAH1The instantaneous of gene is sunk
Silent VIGS carriers, and further transformation of tobacco plant constructs transfer-gen plant, related experiment process is briefly discussed below.
（One）Build instantaneous silence VIGS carriers
First, design PCR amplification primer sequence is as follows：
NtSLAH1-F：5 '-GACGACAAGACCCTGCAGCCTTCTCATCCTCTCTTTTC-3 ',
NtSLAH1-R：5’- TGAGGAGAAGAGCCCTGCAGGCACTCTCTTTCCATTCCT-3’；
PCR amplification is carried out with above-mentioned primer sequence（Amplification length：346bp）Obtain the boot sequence of VIGS；
Secondly, using the method for In-Fusion, by the boot sequence of above-mentioned amplification and TRV carriers（50 DEG C of connection 15min）Connection,
Screening, sequence verification structure obtain and connect correct TRV-NtSLAH1Carrier.
（Two）Convert Agrobacterium
Using freeze-thaw method, by TRV-NtSLAH1After carrier conversion Agrobacterium GV3101, picking positive monoclonal bacterium colony, liquid training
After supporting, ensure that target fragment conversion is correct, and will convert correct bacterium solution and save backup using the verification of bacterium solution PCR method.
It should be noted that as control, under the conditions of similary mode of operation, respectively by TRV1, TRV2, TRV2-PDS（Sun
Property control）It has converted Agrobacterium GV3101 and has been prepared for control transfection bacterium solution.
（Three）Prepare transfection liquid
By step（Three）In prepared contain TRV1, TRV2, TRV2-PDS（Positive control）、TRV2-NtSLAH1Agrobacterium
Single bacterium colony is respectively connected to YEB（5mL）In culture medium (kanamycins, 50 μ g/mL), 28 DEG C, 250 r/min overnight shaking cultures
About 48h；
It is forwarded in the YEB of 50 mL, 28 DEG C of overnight shaking cultures；
4000r/min centrifuge 8 min collect Agrobacterium to 50 mL centrifuge tubes in, and with contain 10 mmol/L 2-N- beautiful jades
The MgCl of base ethanesulfonic acid (MES), 20 μ l/L acetosyringones (Acetosyringone, As) and 10mmol/L2Mixing it is molten
The OD values of above-mentioned bacterium solution are adjusted to 1.0 or so by liquid.
Containing TRV2, TRV2-PDS、TRV-NtSLAH1The medium volume of MMA suspension of Agrobacterium add in containing TRV1
The MMA suspension mixings of Agrobacterium, are placed at room temperature for 3 ~ 6h as transfection liquid.
（Four）It prepares transformant and is converted
By tobacco seed（Ben's tobacco）Nursery in pot for growing seedlings is seeded in, is divided into seedlings within two weeks after germinateing, planted in polypots
（10cm×10cm）In, daily fertilizer and water management etc. is carried out under 22 DEG C, 16h light/8h dark conditions, grows 4 ~ 5w, chooses growing way one
12 basin tobacco seedlings are caused as transformant；
About 4 ~ 5 consistent leaves of growing way are selected during conversion inoculation, are contained with 1mL needle-less asepsis injector by filter press technique handle
There is the agrobacterium suspension of different TRV recombinant plasmids from the blade that vacuum side of blade press-in is all unfolded, make bacterium solution full of entire
Blade is cultivated under 22 DEG C, 75% damp condition；
Wherein, the injection plant of the positive control containing TRV2-PDS is inoculated with 4 basins, remaining empty carrier containing TRV2, containing TRV2-NtSLAH1
Injection plant respectively inoculation 4 basins.
After being inoculated with 6 weeks, detect in each processing group plantNtSLAH1 Gene expression amount（Using real-time PCR skills
Art）And the content of chlorion.
The detection of chloride ion content is carried out with specific reference to following method：
Each processing group plant takes 3 ~ 4 leaves, and 90 DEG C of drying of baking oven are put into after being wrapped up with masking foil overnight；
The sample of drying with beveller is smashed, weighs 0.05g tobacco leaves（It is accurate to 0.0001g）, it is molten to add in 15ml, 5% acetic acid
Liquid is placed in constant temperature oscillator（30 DEG C of shaking tables）, isothermal vibration 30min；
Upper Continuous Flow Analysis instrument measures chloride ion content after filter paper filtering.
It is rightNtSLAH1 Gene expression amount testing result as shown in Fig. 2, figure it is seen thatNtSLAH1 Gene expression
It measures relatively low, showsNtSLAH1 Gene is successfully silenced, and Transgenics are built successfully.
It is as shown in Figure 3 to chloride ion content testing result in plant body.From figure 3, it can be seen that in gene silencing plant
Chloride ion content is approximately the 63% of adjoining tree chloride ion content；That is, after gene silencing, chloride ion content has dropped 37% in plant
Left and right.
It can be seen that based on above-mentioned data resultNtSLAH1Gene and the slow anion channel albumen of the tobacco that it is encoded
NtSLAH1 and chlorion transhipment are highly relevant, can be that good base is established in the cultivation of low chlorine content new product of tobacco using this achievement
Plinth.
Claims (7)
1. the encoding gene of the slow anion channel albumen NtSLAH1 of tobaccoNtSLAH1Gene, which is characterized in that gene C DS sequences
Row include 1107bp bases, and base sequence is as shown in SEQ ID NO.1；Wherein the 270th -615 nucleotide are specificity
Nucleic acid fragment.
2. a kind of PCR amplification is obtained described in claim 1NtSLAH1The method of gene, which is characterized in that the specific steps are：
（1）The total serum IgE of tobacco sample is extracted, and reverse transcription is spare for cDNA；
（2）It is as follows to design amplimer sequence：
F：5 '-CGCGAGCTCGGTACCATGGGGGAAGAAGTTTTTG-3 ',
R：5’-GCTCACCATGGATCCCTAATTACGTTTAGTGAAGT-3’；
With step（1）In prepared cDNA be template, utilize above-mentioned primer carry out PCR amplification.
3. described in claim 1NtSLAH1The slow anion channel albumen NtSLAH1 albumen of tobacco of coded by said gene, feature
It is, which is a kind of ionophorous protein, and related to chlorion transhipment, including 368 amino acid, amino acid sequence is such as
Shown in SEQ ID NO.2.
4. applications of the slow anion channel albumen NtSLAH1 of tobacco described in claim 2 in tobacco, which is characterized in that be used for
Transport chlorion.
5. the encoding gene of the slow anion channel albumen NtSLAH1 of tobacco described in claim 1NtSLAH1Gene is in tobacco
Using, which is characterized in that after the gene silencing, chloride ion content is decreased obviously in plant.
6. utilize the encoding gene of the slow anion channel albumen NtSLAH1 of tobacco described in claim 1NtSLAH1Constructed by gene
Be used for silenceNtSLAH1The instantaneous silence VIGS carriers of gene, which is characterized in that its construction method is：Utilize virus induction
Gene silent technology, with the encoding gene of the slow anion channel albumen NtSLAH1 of the tobaccoNtSLAH1The specificity of gene
The nucleic acid specific fragment is connected on transient expression vector TRV, as boot sequence after conversion, into one by nucleotide fragments
For step through screening, identifying, structure obtains instantaneous silence VIGS carriers：TRV-NtSLAH1。
7. silence to be used for described in claim 6NtSLAH1Application of the instantaneous silence VIGS carriers of gene in tobacco, feature
It is, using transgenic technology, after the VIGS carrier transformation of tobacco plant, for silenceNtSLAH1Gene so that tobacco is slow
Anion channel albumen NtSLAH1 expressing quantities are substantially reduced or even are beyond expression, and chlorion contains in final reduction plant
Amount.
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| CN109337914A (en) * | 2018-11-21 | 2019-02-15 | 中国烟草总公司郑州烟草研究院 | The slow anion channel albumen NtSLAH8 of one tobacco and its application |
| CN109439668A (en) * | 2018-11-13 | 2019-03-08 | 云南省烟草农业科学研究院 | A kind of tobacco chloride ion absorbs gene NtSLAC2 and its cloning process and application |
| CN109517828A (en) * | 2018-11-21 | 2019-03-26 | 中国烟草总公司郑州烟草研究院 | The slow anion channel albumen NtSLAH5 of one tobacco and its application |
| CN113832165A (en) * | 2021-11-08 | 2021-12-24 | 云南省烟草农业科学研究院 | Tobacco NtSLAH3 gene mutant and molecular identification method and application thereof |
| CN113930432A (en) * | 2021-11-08 | 2022-01-14 | 云南省烟草农业科学研究院 | Tobacco NtSLAC1 gene mutant and molecular identification method and application thereof |
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| CN109439668B (en) * | 2018-11-13 | 2021-12-24 | 云南省烟草农业科学研究院 | Tobacco chloride ion absorbing gene NtSLAC2 and cloning method and application thereof |
| CN109337914A (en) * | 2018-11-21 | 2019-02-15 | 中国烟草总公司郑州烟草研究院 | The slow anion channel albumen NtSLAH8 of one tobacco and its application |
| CN109517828A (en) * | 2018-11-21 | 2019-03-26 | 中国烟草总公司郑州烟草研究院 | The slow anion channel albumen NtSLAH5 of one tobacco and its application |
| CN109517828B (en) * | 2018-11-21 | 2022-05-06 | 中国烟草总公司郑州烟草研究院 | Tobacco slow anion channel protein NtSLAH5 and application thereof |
| CN113832165A (en) * | 2021-11-08 | 2021-12-24 | 云南省烟草农业科学研究院 | Tobacco NtSLAH3 gene mutant and molecular identification method and application thereof |
| CN113930432A (en) * | 2021-11-08 | 2022-01-14 | 云南省烟草农业科学研究院 | Tobacco NtSLAC1 gene mutant and molecular identification method and application thereof |
| CN113930432B (en) * | 2021-11-08 | 2023-09-12 | 云南省烟草农业科学研究院 | Tobacco NtSLAC1 gene mutant and molecular identification method and application |
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