CN108841834A - One tobacco chloride channel protein NtCLC2 and its application - Google Patents
One tobacco chloride channel protein NtCLC2 and its application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- 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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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/8251—Amino acid content, e.g. synthetic storage proteins, altering amino acid biosynthesis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Abstract
The invention belongs to transgenic tobacco technical fields, and in particular to a tobacco chloride channel protein NtCLC2 and its apply patent application.Gene C DS sequence includes 2343bp base, and base sequence is as shown in SEQ ID NO.1；Wherein the 1426th -1738 nucleotide are nucleic acid specific fragment.Tobacco chloride channel protein NtCLC2 is the key protein of tobacco chloride ion metabolism, passes through virus induced gene silencing（VIGS）Technology, inhibitNtCLC2After gene expression, chloride ion content is substantially reduced in transgene silencing plant.Based on this, good basis can be established to cultivate low chlorine content new product of tobacco, while also provide the technical support of essence for the stabilization of quality of tobacco and cigarette quality improvement.
Description
Technical field
The invention belongs to transgenic tobacco technical fields, and in particular to a tobacco chloride channel protein NtCLC2 and
It applies patent application.
Background technique
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 will affect to glow when 1% and hold fire, will appear grey black flame-out phenomenon when further above 1%；On the other hand, chloride ion contains in tobacco leaf
It is more to will cause starch accumulation when measuring excessively high, blade is plump and crisp, and hygroscopicity is big, so that color easily deepens when 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 components in Henan
（0.53%-0.65%）Much higher than national mean value（0.26%-0.30%）.In addition, according to Shang Yan group 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 the 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 be promoted 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 existing research it is known that be：For CLC（Chloride
Channel）Protein family has 7 members in arabidopsis, research shows that AtCLCa is during driving nitrate to enter vacuole
It plays a key effect, and AtCLCb is just without this function；Other AtCLC are distributed in other cell sections, AtCLCd and
AtCLCf may play acidification in transhipment Golgi saccule network；The anion that AtCLCe may participate in thylakoid membrane seeps
Permeability；AtCLCg is consistent with AtCLCc function, participates in plant salt stress.Jossier in 2010 et al. is in research arabidopsis CLCc
Shi Faxian（The Arabidopsis vacuolar anion transporter, AtCLCc, is involved in the
Regulation of stomatal movements and contributes to salt tolerance, Plant
Journal, 2010,64 (4):563-576）, AtCLCc is positioned at the guard cell of blade, and mutant strain is to salt density value.But it is total
For body, since CLC family protein function is not fully consistent in the diversification and different plant species of CLC family protein function,
Therefore, only with regard to tobacco bred improvement for, for CLC family protein in tobacco function carry out further investigation can be tobacco bred
Improvement even can establish good theoretical basis and application foundation for other plant improvement.
Summary of the invention
It is an object of that present invention to provide a tobacco chloride channel protein NtCLC2（Chloride channel, CLC）,
Tobacco the chloride channel protein NtCLC2 and Cl of the coded by said gene-Absorption and transport it is related, be based on this function, can be low
The new product of tobacco of chloride ion content lays the foundation.
Details are as follows for the technical solution that the application is taken.
The encoding gene of tobacco chloride channel protein NtCLC2NtCLC2Gene, the gene source is in tobacco
（Nicotiana tabacum）, CDS sequence includes 2343bp base, and base sequence is as shown in SEQ ID NO.1；Wherein
1426th -1738 nucleotide are nucleic acid specific fragment.
Described in PCR amplification obtainsNtCLC2The method of gene specifically refers to as follows：
（1）The total serum IgE of tobacco sample is extracted, and reverse transcription is that cDNA is spare；
（2）It is as follows to design amplimer sequence：
F：5 '-CGCGAGCTCGGTACCATGGAAGATCAAGGTGAT -3 ',
R：5'- GCTCACCATGGATCCCTTGTGATGGACCAAAT -3'；
With step（1）In prepared cDNA be template, utilize above-mentioned primer carry out PCR amplification.
Tobacco chloride channel protein NtCLC2 albumen, is a kind of ionophorous protein, which transports phase with chloride ion
It closes, including 480 amino acid, amino acid sequence is as shown in SEQ ID NO.2.
Application of the tobacco chloride channel protein NtCLC2 in tobacco, for transporting chloride ion.
The encoding gene of the tobacco chloride channel protein NtCLC2NtCLC2Application of the gene in tobacco, by the base
After silencing, chloride ion content is decreased obviously in plant.
Encoding gene for silencing tobacco chloride channel protein NtCLC2NtCLC2The instantaneous silencing VIGS of gene is carried
Body, construction method are：Utilize virus induced gene silencing（VIGS）Technology, with the tobacco chloride channel protein
The encoding gene of NtCLC2NtCLC2The specific nucleotide acid fragment of gene connects the nucleic acid specific fragment as boot sequence
It is connected on transient expression vector TRV, after converting bacillus coli DH 5 alpha, is further screened, identified, building obtains instantaneous silencing
VIGS carrier：TRV-NtCLC2。
The encoding gene for silencing tobacco chloride channel protein NtCLC2NtCLC2The instantaneous silencing of gene
Application of the VIGS carrier in tobacco after the VIGS carrier transformation of tobacco plant, is used for silencing using transgenic technologyNtCLC2Gene, so that tobacco chloride channel protein NtCLC2 protein gene expression amount is substantially reduced or even is beyond expression, most
Chloride ion content in the low plant of final decline.
A method of low chlorine plant variety is cultivated, using transgenic technology, the silencing tobacco chloride ion that is used for is led to
The encoding gene of road albumen NtCLC2NtCLC2The instantaneous silencing VIGS carrier of gene converts plant, and screening, identification obtain wink
When silencing plant；
Or the method using RNAi interference, gene silencing vector is constructed, Nicotiana tabacum is converted, can be obtained by screening, identifying
Genetically modified plants new varieties, in genetically modified plants new varietiesNtCLC2The expression of gene is restricted；The plant to be transformed
Body is common cultivation tobacco.
Tobacco chloride channel protein NtCLC2 in the present invention is the key protein of tobacco chloride ion metabolism, is passed through
Real-time PCR discovery shouldNtCLC2Gene is all expressed in each tissue of tobacco, and is organized in tobacco stem, leaf bud etc.
It expresses relatively high.To further confirm that the protein function, pass through virus induced gene silencing（VIGS）Technology, construct
SilencingNtCLC2The VIGS carrier of gene, has successfully been obtained inhibition after conversionNtCLC2The transgenosis expressed in Ben's tobacco
Silencing plant.Testing result shows relative comparison plant, and chloride ion content is substantially reduced in transgene silencing plant, reduces
At least 35%, i.e.,：Transgene silencing plant obtained possesses the special table that chloride ion content relative comparison plant is substantially reduced
Type, in other words, silencingNtCLC2The intracorporal chloride ion content of plant can be significantly reduced after gene.
It can be seen that in conjunction with existing technique for gene engineering and pass through knockout or silencing using gene silent technologyNtCLC2Base
Because after, it can be substantially reduced the intracorporal chloride ion content of plant, be based on this, can be established for cultivation low chlorine content new product of tobacco good
Good basis, while also the technical support of essence is provided for the stabilization of quality of tobacco and cigarette quality improvement.
Detailed description of the invention
Fig. 1 is in different tissues organNtCLC2Relative expression quantity；
Fig. 2 is in silencing plantNtCLC2 The relative expression quantity of gene；
Fig. 3 is the chloride ion content after drying in each processing group tobacco leaf.
Specific embodiment
Explanation is further explained to the application below with reference to embodiment, before introducing specific embodiment, 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 the raw work in Shanghai；
Experiment reagent：
LA Taq enzyme, PstI restriction enzyme, plasmid extraction kit, plastic recovery kit etc. are purchased from Takara company,
In-Fusion T-A clone kit is purchased from clontech company；
RNA extracts kit is purchased from GeneAnswer company；
Reverse transcription reagent box, RT-PCR kit are purchased from Roche company；
Peptone, yeast extract etc. are purchased from Oxoid company；
Portion of reagent formula and preparation method are briefly described as follows：
（1）LB fluid nutrient medium（1L）：10 g bacto peptones（bacteriological peptone）, 10 g sodium chloride
（NaCl）, 5 g yeast extracts（yeast extract）, autoclave sterilization；
（2）YEB fluid nutrient medium（1L）：5 g beef extracts（beef extract）, 5 g bacto peptones
（bacteriological peptone）, 5 g sucrose（sucrose）, 1 g yeast extract（yeast extract）, 2
Ml 1M magnesium sulfate（MgSO4）, autoclave sterilization；
（3）1M 2- (N- morpholine) ethanesulfonic acid（MES）Stock solution：ddH2O dissolves MES, and filtration sterilization, -20 DEG C store for future use；
（4）200 mM acetosyringones（Acetosyringone）Stock solution：Dimethyl sulfoxide（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 acetosyringone（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 tobacco chloride channel proteinNtCLC2The acquisition process of gene, is briefly discussed below.
Using cultivar tobacco leaf as sample, tobacco leaf total serum IgE is extracted using RNA extracts kit, reverse transcription is
CDNA is spare；
By the method for homologous comparison, with reference to arabidopsisAtCLC2The sequence of gene and known tobacco section gene order, design
Amplimer sequence is as follows：
F：5 '-CGCGAGCTCGGTACCATGGAAGATCAAGGTGAT -3 ',
R：5'- GCTCACCATGGATCCCTTGTGATGGACCAAAT -3'；
Using above-mentioned prepared cDNA as template, PCR amplification is carried out using above-mentioned primer.
PCR reaction system is：
1 μ L of upstream primer,
1 μ L of downstream primer,
1 μ L of cDNA,
10 × buffer, 5 μ L,
6 μ L of dNTP,
1 μ L of EazyTaq enzyme,
Add ddH2O to 50 μ L.
PCR response procedures：94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s, 55 DEG C of annealing 45s, 72 DEG C of extension 1min, 35
Circulation；72 DEG C of extension 10min.
PCR product recycles purpose band after the detection of 1% agarose gel electrophoresis.It will be above-mentioned using In-Fusion method
PCR product is connect with plant expression vector pFF19, converts E. coli competent DH5 α, 37 DEG C of overnight incubations.
It is sequenced after being purified to amplified production, obtains the encoding gene of tobacco chloride channel protein NtCLC2NtCLC2Gene includes 2343bp base altogether, analysis shows wherein the 1426th -1738 nucleotide are specific nucleic acid piece
Section, base sequence are specific as follows as shown in SEQ ID NO.1：
ATGGAAGATCAAGGTGATATAGAGAATGAAGGAGGAGGAATTGGGGTGATGATAATGGAGAATGGCAAAGATT
TGGAGAGGAATATTTCAGCGGTTTCTGAGAGTGGTGTTAGACAGCCATTGCTTAGTTCTAAAAGCAGAGTCAATAAT
ACCTCACAAATTGCTATTATAGGAGCCAATGTTTGCCCCATTGAAAGTCTTGATTATGAGATTATTGAAAATGACCT
TTTCAAGCAAGATTGGAGATCTAGGAAAAAAGTCCAGATATTTCAATATATATTCCTAAAGTGGACACTTGTGCTTC
TTATTGGATTGAGTGTAGGGCTTGTTGGTTTCTTTTTAAACATAGCAGTGGAAAATATTGCTGGCTTCAAGCTTCTG
CTCATTAGTGACTTAATGCTTCAGGACAAGTATTTCCGTGGATTTGCTGCTTATGCATGTTGCAATTTGGTTCTTGC
GACTTGTGCTGGAATCCTATGTGCATTTATTGCACCAGCAGCTGCAGGGTCAGGAATACCTGAAGTGAAAGCATATC
TCAATGGTATCGATGCTCATTCCATTTTAGCTCCAAGTACTTTATTTGTGAAGATATTTGGTTCTGCTTTGGGTGTT
TCTGCTGGATTTGTTGTTGGCAAGGAAGGACCCATGGTCCATACTGGCGCTTGCATAGCAAACTTGCTTGGACAGGG
CGGCTCCCGCAAGTATCATCTGACTTGGAAGTGGCTGAAGTATTTCAAAAATGACCGTGACCGTAGAGATTTGATCA
CTTGTGGTGCTGCAGCTGGTGTTGCAGCTGCTTTCCGAGCCCCAGTCGGTGGTGTTCTTTTTGCTCTTGAAGAAGTA
GCCTCATGGTGGCGAAGTGCTCTTCTTTGGAGGACCTTCTTCTCGACTGCTGTAGTAGCTATGGTGCTCAGATCTTT
CATTGTATTCTGTCGGAGCGGGAAATGTGGACTATTTGGTCAAGGAGGTTTGATAATGTATGATGTGAATTCAGGAG
CACCTAATTATAACACTATAGATGTACTGGCAGTGTTGTTAATTGGAGTTCTTGGAGGCCTTCTCGGAAGCCTTTAT
AATTATCTAGTGGACAAGGTCCTCCGGACTTACAGCATCATTAATGAGAGGGGTCCTGCTTTCAAAGTCTTGCTCGT
AATGACCATTTCAATCCTGAGTTCTCTTTGCTCGTATGGTCTACCGTGGTTTGCAACTTGCACACCATGTCCTGTAG
GCTTGGAGGACAAGTGCCCTACTATAGGTCGCTCTGGAAACTATAAGAATTTCCAGTGTCCAGCGGGGCATTACAAT
GATCTAGCCTCCCTGTTTATGAATACCAATGATGACGCCATCCGCAATTTGTTTAGCTCAGATAATTCCAGCGAATT
TCACCTCTCTTCGCTTTTTGTCTTCTTTGCTGGGGTATATTGCCTTGGCGTCGTTACTTATGGAATTGCTATTCCCT
CTGGGCTATTCATTCCCGTCATACTTGCCGGTGCCTCCTATGGACGTTTTGTTGGGACTGTCTTGGGTTCGATATCC
AATCTTAATAACGGCCTCTTTGCTCTCCTTGGTGCTGCTTCCTTCCTCGGGGGTACTATGAGGATGACGGTATCCAT
CTGTGTCATACTACTTGAGCTCACCGATGACCTGCTAATGCTTCCATTGGTGATGCTTGTGCTCCTTATTTCAAAGA
CTGTGGCTGATTGTTTTAACCATGGTGTCTATGACCAGATTGTGAAAATGAAAGGCTTGCCTTATTTGGAAGCACAC
GCCGAACCATACATGAGGCAATTAGTTGCAGGAGATGTTTGTTCAGGGCCTTTAATTACATTTTCAGGTGTTGAGAA
GGTAGGGAACATAATACATGCTTTGAAGTTTACTAGACACAATGGGTTTCCCGTGATTGATGCACCGCCATTCTCAG
ACGCGCCAGAGTTCTGTGGACTTGCTTTAAGGTCACACCTACTTGTTTTGCTCAAAGCAAAGAAATTCACGAAACTA
AGTGTATTGAGCGGCTCCAGTATTTTGAGGAGTTTTCATGCGTTTGATTTCGCTAAGCCAGGATCGGGGAAGGGGCC
TAAGCTCGAGGATTTGTCCTTCACCGATGAGGAGATGGAAATGTATGTAGATCTCCATCCTGTCACAAATACATCTC
CATACACAGTAGTGGAAACCATGTCTCTGGCCAAAGCTGCAATCCTTTTTCGACAACTTGGTCTGAGACACTTGTGT
GTTGTACCAAAAAAGACTACCGGGAGGGATCCAATAGTTGGAATTTTGACAAGGCATGACTTTATGCCAGAACATAT
AAAGGGACTGTACCCACATTTGGTCCATCACAAGTAG。
To encoding geneNtCLC2After gene is analyzed, is translated, it is known that the ammonia of tobacco chloride channel protein NtCLC2
Base acid sequence, the albumen include 480 amino acid altogether, and amino acid sequence is specific as follows as shown in SEQ ID NO.2：
AMVLRSFIVFCRSGKCGLFGQGGLIMYDVNSGAPNYNTIDVLAVLLIGVLGGLLGSLYNYLVDKVLRTYSIIN
ERGPAFKVLLVMTISILSSLCSYGLPWFATCTPCPVGLEDKCPTIGRSGNYKNFQCPAGHYNDLASLFMNTNDDAIR
NLFSSDNSSEFHLSSLFVFFAGVYCLGVVTYGIAIPSGLFIPVILAGASYGRFVGTVLGSISNLNNGLFALLGAASF
LGGTMRMTVSICVILLELTDDLLMLPLVMLVLLISKTVADCFNHGVYDQIVKMKGLPYLEAHAEPYMRQLVAGDVCS
GPLITFSGVEKVGNIIHALKFTRHNGFPVIDAPPFSDAPEFCGLALRSHLLVLLKAKKFTKLSVLSGSSILRSFHAF
DFAKPGSGKGPKLEDLSFTDEEMEMYVDLHPVTNTSPYTVVETMSLAKAAILFRQLGLRHLCVVPKKTTGRDPIVGI
LTRHDFMPEHIKGLYPHLVHHK。
In order to further appreciate thatNtCLC2Expression specificity of the gene in different tissues utilizes real-time PCR skill
Art is right respectively to cultivate the organs such as cigarette seed, maturity period tobacco leaf, stem, root, leaf bud, stamen, gynoecium and calyx as sampleNtCLC2Expression of the gene in different tissues is detected.As a result as shown in Figure 1.
Analysis can be seen thatNtCLC2Gene has expression in each tissue of tobacco, and in tobacco stem, flower（Flower
Bud, gynoecium, stamen, calyx）Expression quantity highest in equal tissues, this also indicate that the gene and plant nutrient absorption in the early stage and
The growth and breeding level interval in later period is related.
Embodiment 2
For determinationNtCLC2Function of the gene in tobacco, selectionNtCLC2Specific nucleic acid segment in gene（Sequence table SEQ ID
The 1426th -1738 nucleotide sequences of NO.1）As boot sequence, silencing is constructedNtCLC2The instantaneous silencing of gene
With VIGS carrier, and further, transformation of tobacco plant constructs transgenic plant, and related experiment process is briefly discussed below.
（One）Construct instantaneous silencing VIGS carrier
Firstly, design PCR amplification primer sequence is as follows：
NtCLC2-F：5 '-CGACGACAAGACCCTCTTGGCGTCGTTACTTAT-3 ',
NtCLC2-R：5'- GAGGAGAAGAGCCCTTTCACAATCTGGTCATAG-3'；
PCR amplification is carried out with above-mentioned primer sequence（Amplification length：312bp）Obtain the boot sequence of VIGS；
Secondly, using the method for In-Fusion, by the boot sequence of above-mentioned amplification and TRV carrier（50 DEG C of connection 15min）Connection,
Screening, sequence verification building obtain and connect correct TRV-NtCLC2Carrier.
（Two）Convert Agrobacterium
Using freeze-thaw method, by TRV-NtCLC2After carrier converts Agrobacterium GV3101, picking positive monoclonal bacterium colony, Liquid Culture
Afterwards, ensure that target fragment converts correctly using the verifying of bacterium solution PCR method, and correct bacterium solution will be converted and saved backup.
It should be noted that as control, under the conditions of same 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-NtCLC2Agrobacterium
Single colonie 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 be centrifuged 8 min collect Agrobacterium into 50 mL centrifuge tubes, and with contain 10 mmol/L 2-N- beautiful jades
The MgCl of base ethanesulfonic acid (MES), 20 μ l/L acetosyringone (Acetosyringone, As) and 10mmol/L2Mixing it is molten
The OD value of above-mentioned bacterium solution is adjusted to 1.0 or so by liquid.
Containing TRV2, TRV2-PDS、TRV-NtCLC2The medium volume of MMA suspension of Agrobacterium be added and contain TRV1
The MMA suspension of Agrobacterium mixes, and is placed at room temperature for 3 ~ 6h as transfection liquid.
（Four）Preparation transformant is simultaneously converted
By tobacco seed（Ben's tobacco）It is seeded in nursery in pot for growing seedlings, is divided into seedlings within two weeks after germinateing, is planted in polypots
（10cm×10cm）In, daily fertilizer and water management etc. is carried out under 22 DEG C, 16h light/8h dark condition, grows 4 ~ 5w, chooses growing way one
Cause 12 basin tobacco seedlings as transformant；
Consistent about 4 ~ 5 leaves of growing way are selected when 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 indentation is all unfolded, makes 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 contains TRV2-NtCLC2's
Injection plant is respectively inoculated with 4 basins.
After being inoculated with 6 weeks, detect in each processing group plantNtCLC2 Gene expression amount（Using real-time PCR technology）
And the content of chloride ion.
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 baking oven drying are put into after being wrapped up with masking foil overnight；
The sample of drying is smashed with beveller, weighs 0.05g tobacco leaf（It is accurate to 0.0001g）, be added 15ml, 5% acetic acid it is molten
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 rightNtCLC2 Gene expression amount testing result as shown in Fig. 2, figure it is seen thatNtCLC2 Gene expression amount
It is lower, showNtCLC2 Gene is successfully silenced, and Transgenics construct 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, chloride ion content has dropped 37% in plant after gene silencing
Left and right.
It can be seen that based on above-mentioned data resultNtCLC2The tobacco chloride channel protein NtCLC2 that gene is encoded with it
It is highly relevant with chloride ion transhipment, using this achievement, good basis can be established for the cultivation of low chlorine content new product of tobacco.
SEQUENCE LISTING
<110>Zhengzhou Tobacco Research Institute of CNTC
<120>One tobacco chloride channel protein NtCLC2 and its application
<130> none
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 2343
<212> DNA
<213> Nicotiana tabacum
<400> 1
atggaagatc aaggtgatat agagaatgaa ggaggaggaa ttggggtgat gataatggag 60
aatggcaaag atttggagag gaatatttca gcggtttctg agagtggtgt tagacagcca 120
ttgcttagtt ctaaaagcag agtcaataat acctcacaaa ttgctattat aggagccaat 180
gtttgcccca ttgaaagtct tgattatgag attattgaaa atgacctttt caagcaagat 240
tggagatcta ggaaaaaagt ccagatattt caatatatat tcctaaagtg gacacttgtg 300
cttcttattg gattgagtgt agggcttgtt ggtttctttt taaacatagc agtggaaaat 360
attgctggct tcaagcttct gctcattagt gacttaatgc ttcaggacaa gtatttccgt 420
ggatttgctg cttatgcatg ttgcaatttg gttcttgcga cttgtgctgg aatcctatgt 480
gcatttattg caccagcagc tgcagggtca ggaatacctg aagtgaaagc atatctcaat 540
ggtatcgatg ctcattccat tttagctcca agtactttat ttgtgaagat atttggttct 600
gctttgggtg tttctgctgg atttgttgtt ggcaaggaag gacccatggt ccatactggc 660
gcttgcatag caaacttgct tggacagggc ggctcccgca agtatcatct gacttggaag 720
tggctgaagt atttcaaaaa tgaccgtgac cgtagagatt tgatcacttg tggtgctgca 780
gctggtgttg cagctgcttt ccgagcccca gtcggtggtg ttctttttgc tcttgaagaa 840
gtagcctcat ggtggcgaag tgctcttctt tggaggacct tcttctcgac tgctgtagta 900
gctatggtgc tcagatcttt cattgtattc tgtcggagcg ggaaatgtgg actatttggt 960
caaggaggtt tgataatgta tgatgtgaat tcaggagcac ctaattataa cactatagat 1020
gtactggcag tgttgttaat tggagttctt ggaggccttc tcggaagcct ttataattat 1080
ctagtggaca aggtcctccg gacttacagc atcattaatg agaggggtcc tgctttcaaa 1140
gtcttgctcg taatgaccat ttcaatcctg agttctcttt gctcgtatgg tctaccgtgg 1200
tttgcaactt gcacaccatg tcctgtaggc ttggaggaca agtgccctac tataggtcgc 1260
tctggaaact ataagaattt ccagtgtcca gcggggcatt acaatgatct agcctccctg 1320
tttatgaata ccaatgatga cgccatccgc aatttgttta gctcagataa ttccagcgaa 1380
tttcacctct cttcgctttt tgtcttcttt gctggggtat attgccttgg cgtcgttact 1440
tatggaattg ctattccctc tgggctattc attcccgtca tacttgccgg tgcctcctat 1500
ggacgttttg ttgggactgt cttgggttcg atatccaatc ttaataacgg cctctttgct 1560
ctccttggtg ctgcttcctt cctcgggggt actatgagga tgacggtatc catctgtgtc 1620
atactacttg agctcaccga tgacctgcta atgcttccat tggtgatgct tgtgctcctt 1680
atttcaaaga ctgtggctga ttgttttaac catggtgtct atgaccagat tgtgaaaatg 1740
aaaggcttgc cttatttgga agcacacgcc gaaccataca tgaggcaatt agttgcagga 1800
gatgtttgtt cagggccttt aattacattt tcaggtgttg agaaggtagg gaacataata 1860
catgctttga agtttactag acacaatggg tttcccgtga ttgatgcacc gccattctca 1920
gacgcgccag agttctgtgg acttgcttta aggtcacacc tacttgtttt gctcaaagca 1980
aagaaattca cgaaactaag tgtattgagc ggctccagta ttttgaggag ttttcatgcg 2040
tttgatttcg ctaagccagg atcggggaag gggcctaagc tcgaggattt gtccttcacc 2100
gatgaggaga tggaaatgta tgtagatctc catcctgtca caaatacatc tccatacaca 2160
gtagtggaaa ccatgtctct ggccaaagct gcaatccttt ttcgacaact tggtctgaga 2220
cacttgtgtg ttgtaccaaa aaagactacc gggagggatc caatagttgg aattttgaca 2280
aggcatgact ttatgccaga acatataaag ggactgtacc cacatttggt ccatcacaag 2340
tag 2343
<210> 2
<211> 480
<212> PRT
<213> Nicotiana tabacum
<400> 2
Ala Met Val Leu Arg Ser Phe Ile Val Phe Cys Arg Ser Gly Lys Cys
1 5 10 15
Gly Leu Phe Gly Gln Gly Gly Leu Ile Met Tyr Asp Val Asn Ser Gly
20 25 30
Ala Pro Asn Tyr Asn Thr Ile Asp Val Leu Ala Val Leu Leu Ile Gly
35 40 45
Val Leu Gly Gly Leu Leu Gly Ser Leu Tyr Asn Tyr Leu Val Asp Lys
50 55 60
Val Leu Arg Thr Tyr Ser Ile Ile Asn Glu Arg Gly Pro Ala Phe Lys
65 70 75 80
Val Leu Leu Val Met Thr Ile Ser Ile Leu Ser Ser Leu Cys Ser Tyr
85 90 95
Gly Leu Pro Trp Phe Ala Thr Cys Thr Pro Cys Pro Val Gly Leu Glu
100 105 110
Asp Lys Cys Pro Thr Ile Gly Arg Ser Gly Asn Tyr Lys Asn Phe Gln
115 120 125
Cys Pro Ala Gly His Tyr Asn Asp Leu Ala Ser Leu Phe Met Asn Thr
130 135 140
Asn Asp Asp Ala Ile Arg Asn Leu Phe Ser Ser Asp Asn Ser Ser Glu
145 150 155 160
Phe His Leu Ser Ser Leu Phe Val Phe Phe Ala Gly Val Tyr Cys Leu
165 170 175
Gly Val Val Thr Tyr Gly Ile Ala Ile Pro Ser Gly Leu Phe Ile Pro
180 185 190
Val Ile Leu Ala Gly Ala Ser Tyr Gly Arg Phe Val Gly Thr Val Leu
195 200 205
Gly Ser Ile Ser Asn Leu Asn Asn Gly Leu Phe Ala Leu Leu Gly Ala
210 215 220
Ala Ser Phe Leu Gly Gly Thr Met Arg Met Thr Val Ser Ile Cys Val
225 230 235 240
Ile Leu Leu Glu Leu Thr Asp Asp Leu Leu Met Leu Pro Leu Val Met
245 250 255
Leu Val Leu Leu Ile Ser Lys Thr Val Ala Asp Cys Phe Asn His Gly
260 265 270
Val Tyr Asp Gln Ile Val Lys Met Lys Gly Leu Pro Tyr Leu Glu Ala
275 280 285
His Ala Glu Pro Tyr Met Arg Gln Leu Val Ala Gly Asp Val Cys Ser
290 295 300
Gly Pro Leu Ile Thr Phe Ser Gly Val Glu Lys Val Gly Asn Ile Ile
305 310 315 320
His Ala Leu Lys Phe Thr Arg His Asn Gly Phe Pro Val Ile Asp Ala
325 330 335
Pro Pro Phe Ser Asp Ala Pro Glu Phe Cys Gly Leu Ala Leu Arg Ser
340 345 350
His Leu Leu Val Leu Leu Lys Ala Lys Lys Phe Thr Lys Leu Ser Val
355 360 365
Leu Ser Gly Ser Ser Ile Leu Arg Ser Phe His Ala Phe Asp Phe Ala
370 375 380
Lys Pro Gly Ser Gly Lys Gly Pro Lys Leu Glu Asp Leu Ser Phe Thr
385 390 395 400
Asp Glu Glu Met Glu Met Tyr Val Asp Leu His Pro Val Thr Asn Thr
405 410 415
Ser Pro Tyr Thr Val Val Glu Thr Met Ser Leu Ala Lys Ala Ala Ile
420 425 430
Leu Phe Arg Gln Leu Gly Leu Arg His Leu Cys Val Val Pro Lys Lys
435 440 445
Thr Thr Gly Arg Asp Pro Ile Val Gly Ile Leu Thr Arg His Asp Phe
450 455 460
Met Pro Glu His Ile Lys Gly Leu Tyr Pro His Leu Val His His Lys
465 470 475 480
Claims (7)
1. the encoding gene of tobacco chloride channel protein NtCLC2NtCLC2Gene, which is characterized in that gene C DS sequence packet
2343bp base is included, base sequence is as shown in SEQ ID NO.1；Wherein the 1426th -1738 nucleotide are specific core
Acid fragment.
2. a kind of PCR amplification obtains described in claim 1NtCLC2The 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 that cDNA is spare；
（2）It is as follows to design amplimer sequence：
F：5 '-CGCGAGCTCGGTACCATGGAAGATCAAGGTGAT-3 ',
R：5'-GCTCACCATGGATCCCTTGTGATGGACCAAAT-3'；
With step（1）In prepared cDNA be template, utilize above-mentioned primer carry out PCR amplification.
3. described in claim 1NtCLC2The tobacco chloride channel protein NtCLC2 albumen of coded by said gene, which is characterized in that
The albumen is a kind of ionophorous protein, related to chloride ion transhipment, including 480 amino acid, amino acid sequence such as SEQ
Shown in ID NO.2.
4. application of the tobacco chloride channel protein NtCLC2 in tobacco described in claim 2, which is characterized in that for transporting
Chloride ion.
5. the encoding gene of tobacco chloride channel protein NtCLC2 described in claim 1NtCLC2Gene answering in tobacco
With, which is characterized in that after the gene silencing, chloride ion content is decreased obviously in plant.
6. utilizing the encoding gene of tobacco chloride channel protein NtCLC2 described in claim 1NtCLC2Use constructed by gene
In silencingNtCLC2The instantaneous silencing VIGS carrier of gene, which is characterized in that its construction method is：Utilize the gene of virus induction
Silent technology, with the encoding gene of the tobacco chloride channel protein NtCLC2NtCLC2The specific nucleotide acid fragment of gene
As boot sequence, which is connected on transient expression vector TRV, after conversion, further screened, reflected
Fixed, building obtains instantaneous silencing VIGS carrier：TRV-NtCLC2。
7. being used for silencing described in claim 6NtCLC2Application of the instantaneous silencing VIGS carrier of gene in tobacco, feature
It is, using transgenic technology, after the VIGS carrier transformation of tobacco plant, is used for silencingNtCLC2Gene, so that tobacco chlorine
Ionophorous protein NtCLC2 expressing quantity is substantially reduced or even is beyond expression, final to reduce chloride ion content in plant.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113136389A (en) * | 2021-04-16 | 2021-07-20 | 河南农业大学 | Genetic engineering application of gene GhCLcg-1A and/or gene GhCLcg-1D |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101054586A (en) * | 2007-03-29 | 2007-10-17 | 上海大学 | Rice chlorine ion passage gene OsCLC, coding protein and clone method thereof |
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2018
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101054586A (en) * | 2007-03-29 | 2007-10-17 | 上海大学 | Rice chlorine ion passage gene OsCLC, coding protein and clone method thereof |
Non-Patent Citations (4)
| Title |
|---|
| ALEXIS DE ANGELI 等: "CLC-mediated anion transport in plant cells", 《PHILOSOPHICAL TRANSACTIONS OF THE ROYAL BSOCIETY》 * |
| NM_001325489.1: "Nicotiana tabacum chloride channel protein CLC-c-like (LOC107790143), mRNA", 《NCBI_GENBANK》 * |
| TAK-HONG WONG 等: "The GmCLC1 protein from soybean functions as a chloride ion transporter", 《JOURNAL OF PLANT PHYSIOLOGY》 * |
| 张惠展: "《基因工程》", 31 January 2017, 华东理工大学出版社 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113136389A (en) * | 2021-04-16 | 2021-07-20 | 河南农业大学 | Genetic engineering application of gene GhCLcg-1A and/or gene GhCLcg-1D |
| CN113136389B (en) * | 2021-04-16 | 2023-02-14 | 河南农业大学 | Genetic engineering application of gene GhCLcg-1A and/or GhCLcg-1D |
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