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CN108192896B - Tobacco slow anion channel protein NtSLAH1 and application thereof - Google Patents

Tobacco slow anion channel protein NtSLAH1 and application thereof Download PDF

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CN108192896B
CN108192896B CN201810137103.0A CN201810137103A CN108192896B CN 108192896 B CN108192896 B CN 108192896B CN 201810137103 A CN201810137103 A CN 201810137103A CN 108192896 B CN108192896 B CN 108192896B
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ntslah1
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张慧
徐国云
金立锋
周会娜
翟妞
刘萍萍
郑庆霞
陈霞
陈千思
王燃
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Zhengzhou Tobacco Research Institute of CNTC
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Abstract

The invention belongs to the technical field of tobacco genetic engineering, and particularly relates to a tobacco slow anion channel protein NtSLAH1 and an application patent application thereof. The CDS sequence of the gene comprises 1107bp bases, and the base sequence is shown as SEQ ID NO. 1; wherein the 270 th to 615 th nucleotides are specific nucleic acid fragments. The tobacco slow anion channel protein NtSLAH1 is a key protein for tobacco chloride ion metabolism, and inhibits the tobacco chloride ion metabolism by the technology of virus-induced gene silencing (VIGS)NtSLAH1After gene expression, the content of chloride ions in transgenic silent plants is obviously reduced. Based on the method, a good foundation can be laid for cultivating new varieties of low-chlorine content tobacco, and meanwhile, fundamental technical support is provided for stabilizing the quality of tobacco leaves and improving the quality of cigarettes.

Description

Tobacco slow anion channel protein NtSLAH1 and application thereof
Technical Field
The invention belongs to the technical field of tobacco genetic engineering, and particularly relates to a tobacco slow anion channel protein NtSLAH1 and an application patent application thereof.
Background
The existing research aiming at tobacco generally considers that the content of chloride ions in tobacco leaves is preferably 0.3-0.8, smoldering fire-holding property can be influenced when the content reaches 1%, and black ash flameout phenomenon can occur when the content is further higher than 1%; on the other hand, when the content of chloride ions in the tobacco leaves is too high, the starch is accumulated more, the leaves are thick and crisp, and the moisture absorption is high, so that the color is easy to become dark during storage, and bad smell is generated. In conclusion, the chloride ion content in tobacco leaves has a relatively important direct influence on the quality of the tobacco leaves.
Based on the direct influence and importance of the content of chloride ions in tobacco leaves on the quality of tobacco, some researchers carry out detection statistics on the content of chloride ions in tobacco leaves all over the country, and the results show that (Zhengzhou tobacco institute, Chinese white paper for tobacco quality (2015)): the content of chloride ions (0.53% -0.65%) in the chemical components of flue-cured tobacco leaves in 2011-2015 of Henan is far higher than the national average (0.26% -0.30%). In addition, according to the average value of the chlorine content determination of the Yuzhong smoke region 2012-2015 by the Shanghai group, especially the 2014 data (2.21% of the lower leaf, 2.09% of the middle leaf and 2.03% of the upper leaf), the chlorine ion content of the Yuzhong smoke region in recent years is far higher than the national level. The statistical data show that the heterogeneity of the tobacco leaf quality in some regions, especially the heterogeneity of the chloride ion content in the tobacco leaf, even higher characteristics, become one of the bottlenecks in restricting the improvement of the quality of the strong aromatic tobacco leaf.
In order to solve the problem of high content of chloride ions in tobacco leaves, the traditional improvement mode mostly starts from a cultivation technology, and the quality of the tobacco leaves is stabilized and improved by optimizing field management measures and perfecting a modulation fermentation technology. However, in general, these measures do not fundamentally change the situation that the quality of the tobacco leaves is low and the industrial applicability is not strong. Thus making the related improvement measures less practical.
With the rapid development of genomics, particularly gene editing technology, people can more and more deeply know the relationship between chloride ion accumulation and related genes in tobacco leaves. Based on prior studies it is already known that: the slow anion channel (SLAC) protein family plays an important role mainly in the uptake of plant anions (chloride, nitrate, etc.) and in the process of opening and closing pores. But because ofSLACThe gene family has more genes and has larger difference in functions in different plants, thereby being suitable for different plants and different plantsSLACThe functions of the genes need to be further researched and distinguished, so that the genes can be used for future plant improvement in a targeted manner.
Disclosure of Invention
The invention aims to provide a tobacco slow Anion Channel protein NtSLAH1 (SLAH activating and Channel Homologue), a tobacco slow Anion Channel protein NtSLAH1 and Cl encoded by the gene-The absorption and the transportation are related, and based on the function, a foundation can be laid for a new variety of tobacco with low chloride ion content.
The technical solution adopted in the present application is detailed as follows.
Coding gene of tobacco slow anion channel protein NtSLAH1NtSLAH1A gene derived from tobacco (A)Nicotiana tabacum) The CDS sequence comprises 1107bp base, and the base sequence is shown as SEQ ID NO. 1; wherein the 270 th to 615 th nucleotides are specific nucleic acid fragments.
Obtained by PCR amplificationNtSLAH1The gene method can be specifically referred to as follows:
(1) extracting total RNA of the tobacco sample, and performing reverse transcription on the total RNA into cDNA for later use;
(2) the amplification primer sequences were designed as follows:
F：5’-CGCGAGCTCGGTACCATGGGGGAAGAAGTTTTTG-3’，
R：5’-GCTCACCATGGATCCCTAATTACGTTTAGTGAAGT-3’；
and (2) taking the cDNA prepared in the step (1) as a template, and performing PCR amplification by using the primer.
The tobacco slow anion channel protein NtSLAH1 is an ion channel protein, is related to chloride ion transport, and comprises 368 amino acids, and the amino acid sequence of the protein is shown in SEQ ID NO. 2.
The application of the tobacco slow anion channel protein NtSLAH1 in tobacco for transporting chloride ions.
The coding gene of the tobacco slow anion channel protein NtSLAH1NtSLAH1The application of the gene in tobacco leads the content of chloride ions in plants to be obviously reduced after the gene is silenced.
Coding gene for silencing tobacco slow anion channel protein NtSLAH1NtSLAH1The construction method of the VIGS vector for transient silencing of the gene comprises the following steps: the coding gene of the tobacco slow anion channel protein NtSLAH1 by using virus-induced gene silencing (VIGS) technologyNtSLAH1The specific nucleotide fragment of the gene is used as a guide sequence, the specific nucleotide fragment is connected to a transient expression vector TRV, and after escherichia coli DH5 alpha is transformed, the transient silencing VIGS vector is constructed by further screening and identifying: TRV-NtSLAH1。
The coding gene for silencing tobacco slow anion channel protein NtSLAH1NtSLAH1The application of VIGS vector for instantly silencing gene in tobacco uses transgenic technology to transform the VIGS vector into tobacco plant for silencingNtSLAH1The gene can obviously reduce the expression level of the tobacco slow anion channel protein NtSLAH1 gene, even can not express the gene, and finally reduce the content of chloride ions in plants.
A method for cultivating low-chlorine plant varieties utilizes a transgenic technology to silence coding genes of tobacco slow anion channel protein NtSLAH1NtSLAH1Transforming plant bodies by the VIGS vector for instantly silencing the gene, and screening and identifying to obtain instantly silencing plants; further, a gene silencing vector is constructed by using an RNAi interference method, common tobacco is transformed, and a transgenic plant can be obtained through screening and identificationNovel variety, novel variety of transgenic plantNtSLAH1The expression of the gene is restricted; the plant body to be transformed is the tobacco cultivated in the common.
The tobacco slow anion channel protein NtSLAH1 is a key protein for tobacco chloride ion metabolism, and is discovered by real-time PCRNtSLAH1The gene is expressed in all tissues of tobacco, and the expression is relatively high in tobacco roots. To further confirm the function of the protein, silencing was constructed by the technique of virus-induced gene silencing (VIGS)NtSLAH1The VIGS vector of the gene successfully obtains inhibition after transformationNtSLAH1A transgenic silent plant expressed in Nicotiana benthamiana. The detection result shows that compared with a control plant, the content of chloride ions in the transgenic silent plant is obviously reduced by at least 35 percent, namely: the obtained transgenic silent plant has a specific phenotype that the content of chloride ions is obviously reduced relative to that of a control plant, in other words, the silent plantNtSLAH1The content of chloride ions in plants can be obviously reduced after the gene is used.
As can be seen by combining the existing gene engineering techniques, gene silencing techniques are utilized to achieve the desired level of gene silencing by knock-out or silencingNtSLAH1After the gene is expressed, the content of chloride ions in the plant body can be obviously reduced, so that a good foundation can be laid for cultivating a new variety of tobacco with low chloride content, and a fundamental technical support is provided for stabilizing the quality of tobacco leaves and improving the quality of cigarettes.
Drawings
FIG. 1 shows different tissues and organsNtSLAH1The relative expression level of (3);
FIG. 2 shows silencing in plantsNtSLAH1 Relative expression level of the gene;
FIG. 3 shows the chloride ion content in tobacco leaves of each treatment group after drying.
Detailed Description
The present application is further illustrated by the following examples, and prior to describing the specific examples, the basic aspects of the biological materials, reagents, instruments, etc. involved in the examples described below are briefly described as follows.
Biological material:
tobacco material: ben's diseaseTobacco (A)Nicotiana benthamiana) A commercial tobacco variety;
interference vector: TRV, purchased from China plasmid vector bacterial cell gene collection center;
gene sequencing and primer synthesis are completed by Shanghai worker;
experimental reagent:
LA Taq enzyme, PstI restriction enzyme, plasmid extraction kit, gel recovery kit, etc., purchased from Takara,
an In-Fusion one-step cloning kit purchased from clontech;
an RNA extraction kit purchased from GeneAnswer;
a reverse transcription kit and an RT-PCR kit purchased from Roche company;
peptone, yeast extract, etc. available from Oxoid corporation;
the formula and preparation method of part of the reagents are briefly described as follows:
(1) LB liquid medium (1L): 10 g bacterial peptone (bacteriological peptone), 10 g sodium chloride (NaCl), 5g yeast extract (yeast extract), autoclaving;
(2) YEB liquid medium (1L): 5g beef extract (beef extract), 5g bacterial peptone (bacterial peptone), 5g sucrose (sucrose), 1g yeast extract (yeast extract), 2 ml 1M magnesium sulfate (MgSO 4), autoclaving;
(3) 1M 2- (N-morpholine) ethanesulfonic acid (MES) stock: ddH2Dissolving MES in O, filtering, sterilizing, and storing at-20 deg.C;
(4) 200 mM Acetosyringone (Acetosyringone) stock solution: dissolving acetosyringone in Dimethyl Sulfoxide (DSMO), and storing at-20 deg.C;
(5) MMA (1L): 20 g sucrose (sucrose), 5g MS salts (Duchefa Biochemie), 1.95 g MES, 1ml acetosyringone (200 mM, pH = 5.6;
an experimental instrument:
PCR apparatus Tgradient, product of Biometra,
real-time quantitative PCR Instrument LightCycler 96, product of Roche.
Example 1
This example is mainly for tobacco slow anion channel proteinsNtSLAH1The process of obtaining the gene is briefly described below.
Taking cultivated species tobacco leaves as a sample, extracting total RNA of the tobacco leaves by using an RNA extraction kit, and performing reverse transcription to obtain cDNA for later use;
method by homology alignment, reference ArabidopsisAtSLAH1The sequence of the gene and the known partial gene sequence of the tobacco are as follows:
F：5’-CGCGAGCTCGGTACCATGGGGGAAGAAGTTTTTG-3’，
R：5’-GCTCACCATGGATCCCTAATTACGTTTAGTGAAGT-3’；
the prepared cDNA is used as a template, and the primer is used for PCR amplification.
During PCR amplification, the reference design of a 50-L reaction system is as follows:
upstream primer F, 1 μ L;
downstream primer R, 1 μ L;
cDNA template, 1. mu.L;
10×buffer，5μL；
dNTP ，6μL；
EazyTaq enzyme, 1 μ L;
add ddH2O to 50 μ L;
PCR reaction procedure: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30s, annealing at 55 ℃ for 45s, extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 10 min.
After the PCR product was detected by 1% agarose gel electrophoresis, the band of interest was recovered. Further, the purified PCR product was ligated with a plant expression vector pFF19 by In-Fusion method, transformed into E.coli competent DH 5. alpha. and cultured overnight at 37 ℃. After screening and identification, the plasmid with correct transformation is purified and sequenced to obtain the coding gene of the tobacco slow anion channel protein NtSLAH1NtSLAH1The gene comprises 1107bp bases, and analysis shows that the 270 th-615 th nucleotides are specific nucleic acid fragments and basesThe base sequence is shown as SEQ ID NO.1, and specifically comprises the following steps:
ATGGGGGAAGAAGTTTTTGAATCAACAATCAAAGTCACAATAAGTGATGATACTATGAAATCAAATGTTGCCAAGAAATCATCTTCTGCTTCTCTTTTGACTAAACTACATGCAGGCTATTTCAGAATAAGCCTATCTTTAGGTGGTCAAGCCTTGTTATGGAAAGTTCTAATTGAACATTTAGATAAATCACAAACTCTTCACCACTTATTTCATACTCTCCCTTCAACTACTTTCCTCTTACTATGGTGGATTTCCCTTTGTACCCTTCTCATCCTCTCTTTTCTTTACATTTTAAGGTGCATTTTTCACTTCTCATTAGTTAAATCAGAGTTTTTACATCCTGTTGGTGTAAACTATCTCTTTGCTCCTTGGATTTCTTGGCTTTTATTACTCCAATCAGCACCATTTAGTATTCCACATGTTGGTTCTTGCCAAGTTTTTTGGTGGATTTTCGTCGTTCCGGTTGGGATTCTTGATGTGAAAATATATGGACAATGGTTTACTACTGAGAAAAGGTTTTTATCAATGGTTGCAAATCCAACTAGCCAACTTTCTGTGTTGGGAAATTTGACTGGTGCTTGGGTTGCAAGTAAAAAGGAATGGAAAGAGAGTGCTGTTTGTATATTTACATTAGGGTTAACACATTATTTGGTAGTGTTTATTACACTTTATCAAAGATTATCTGGTAGTAATAACCTACCTGCTATGCTTAGACCTTCTTTCTTTTTGTTTGTGGCTGCTCCTAGTATGGCTAGCTTAGCTTGGGCTTCTATTTCTGGGAATTTTGATATGTCATGCAGAATGCTCTTTTTTCTCTCACTATTTCTCTTCACTTCTTTGGTTTGTAGGCCAGCACTATTCAAGAAATCAATGAGAAAGTTCAATGTTGCATGGTGGGCTTACTCATTTCCTCTCACATTCCTAGCCTTAGCCTCTGCACAATATGCACATCAAGTGAAAGGTCCTGTTTCTGCTGGACTTATGCTGCTTCTCTCAGCCCTTTCAGTTCTTGTTTTTGTTGGTTTGACAGTTTCCACTGCTCTCAATCTTGACATGCTTTTGGCTGATAATGATCGCTATTTAAACTTCACTAAACGTAATTAG。
for coding geneNtSLAH1After the gene is analyzed and translated, the amino acid sequence of the tobacco slow anion channel protein NtSLAH1 can be known, the protein totally comprises 368 amino acids, and the amino acid sequence is shown as SEQ ID NO.2, and specifically comprises the following steps:
MGEEVFESTIKVTISDDTMKLDVAKKPSSASLLTKLHAGYFRISLSLGGQALLWKVLIEHLDKSKTLHHLFHTLPSTTFLLLWWISLCTLIILSFLYILRCIFHFSLVKSEFLHPVGVNYLFAPWISWLLLLQSAPFSIPHVGSCQVFWWIFVIPVGILDVKIYGQWFTTEKRFLSMVANPTSQLSVLGNLTGAWIASKKEWKESAVCIFTLGLTHYLVVFITLYQRLSGSNSLPAMLRPSFFLFVAAPSMASLAWASISGDFDMSCRMLFFLSLFLFTSLVCRPAIFKKSMKKFNVAWWSYSFPLTFLALASVQYAHQVKSPVSAGLMLLLSALSVLVFVGLTVSTALNLDMLLADNDRYLNFTKRN。
to further understandNtSLAH1The expression specificity of the gene in different tissues is realized by respectively cultivating tobacco seeds, tobacco leaves in the mature period and the like by using real-time PCR technology,Organs such as stem, root, leaf bud, stamen, pistil and calyx, etc. as samples, andNtSLAH1the expression of the gene in different tissues was examined. The results are shown in FIG. 1.
As can be seen from the analysis,NtSLAH1the gene is expressed in each tissue of tobacco, but the expression level is highest in the root of tobacco.
Example 2
To determineNtSLAH1Function of genes in tobacco, selectionNtSLAH1A specific nucleic acid fragment (nucleotide sequence 270-615 th site of SEQ ID NO.1 of a sequence table) in the gene is used as a guide sequence to construct silenceNtSLAH1Transient silencing for genes uses a VIGS vector, and further transforms tobacco plants to construct transgenic plants, and related experimental processes are briefly introduced as follows.
Construction of VIGS vectors for transient silencing
First, primer sequences for PCR amplification were designed as follows:
NtSLAH1-F：5’- GACGACAAGACCCTGCAGCCTTCTCATCCTCTCTTTTC-3’，
NtSLAH1-R：5’- TGAGGAGAAGAGCCCTGCAGGCACTCTCTTTCCATTCCT-3’；
PCR amplification (amplification length: 346 bp) is carried out by the primer sequence to obtain a VIGS guide sequence;
secondly, the amplified guide sequence is connected with a TRV vector (at 50 ℃ for 15 min) by using an In-Fusion method, and the TRV-plus with correct connection is obtained by screening, sequencing and verifying constructionNtSLAH1And (3) a carrier.
(II) transformation of Agrobacterium
TRV is treated by freeze-thawingNtSLAH1After the vector transforms agrobacterium GV3101, a positive monoclonal colony is selected, after liquid culture, a bacterial liquid PCR method is used for verification and ensuring that the target fragment is correctly transformed, and the bacterial liquid with the correct transformation is stored for later use.
As a control, Agrobacterium GV3101 was transformed with TRV1, TRV2, and TRV2-PDS (positive control) under the same operation conditions, and a control transfection solution was prepared.
(III) preparation of transfection solution
The fraction prepared in the step (three) containing TRV1, TRV2, TRV2-PDS (positive control), TRV2-NtSLAH1Inoculating the single colonies of the agrobacterium into YEB (5 mL) culture medium (kanamycin, 50 mu g/mL) respectively, and carrying out shaking culture at 28 ℃ and 250 r/min overnight for about 48 h;
transferring to 50 mL YEB, and shaking and culturing at 28 ℃ overnight;
the Agrobacterium was collected by centrifugation at 4000r/min for 8 min into a 50 mL centrifuge tube and used with a solution containing 10 mmol/L2-N-morpholinoethanesulfonic acid (MES), 20. mu.l/L Acetosyringone (Acetosyringone, As) and 10mmol/L MgCl2The OD value of the bacterial suspension was adjusted to about 1.0.
In the case of the alloy containing TRV2, TRV2-PDS、TRV-NtSLAH1Adding MMA suspension containing TRV1 agrobacterium into the MMA suspension of the agrobacterium in the same volume, mixing uniformly, and standing at room temperature for 3-6 h to serve as transfection solution.
(IV) preparation of transformants and transformation
Sowing tobacco seeds (Nicotiana benthamiana) in a seedling pot for seedling cultivation, dividing seedlings two weeks after germination, planting the seedlings in a plastic pot (10 cm multiplied by 10 cm), performing daily fertilizer and water management and the like under the dark condition of 16h light/8 h at the temperature of 22 ℃, growing for 4-5 w, and selecting 12 seedlings with consistent growth vigor as transformants;
selecting about 4-5 leaves with consistent growth vigor during transformation and inoculation, pressing agrobacterium tumefaciens suspension liquid containing different TRV recombinant plasmids into all unfolded leaves from the back of the leaves by a 1mL pinless sterile injector through a filter pressing method so that the whole leaves are filled with the bacterium liquid, and culturing under the conditions of 22 ℃ and 75% humidity;
wherein, the injection plant containing TRV2-PDS positive control is inoculated into 4 pots, and the rest contains TRV2 empty vector and TRV2-NtSLAH1The injected plants of (2) were inoculated into 4 pots each.
6 weeks after inoculation, plants in each treatment group were testedNtSLAH1 Gene expression level (using real-time PCR technology) and chloride ion content.
The detection of the content of the chloride ions is carried out by specifically referring to the following method:
taking 3-4 leaves from each treatment group, wrapping the leaves with tinfoil paper, and drying the leaves in an oven at 90 ℃ overnight;
crushing the dried sample with a grinder, weighing 0.05g tobacco (accurate to 0.0001 g), adding 15ml 5% acetic acid solution, placing in a constant temperature oscillator (30 deg.C shaking table), and shaking at constant temperature for 30 min;
and filtering the filter paper, and then measuring the content of the chloride ions by using a continuous flow analyzer.
To pairNtSLAH1 The results of the gene expression level measurement are shown in FIG. 2, and it can be seen from FIG. 2,NtSLAH1 lower gene expression level, indicating thatNtSLAH1 The gene is successfully silenced, and the transgene body is successfully constructed.
The results of detecting the chloride ion content in the plant are shown in FIG. 3. As can be seen in FIG. 3, the chloride content in the gene-silenced plants is about 63% of the chloride content of the control plants; namely, after gene silencing, the content of chloride ions in plants is reduced by about 37%.
As can be seen from the above data results,NtSLAH1the gene and the tobacco slow anion channel protein NtSLAH1 coded by the gene are highly related to chloride ion transport, and by utilizing the result, a good foundation can be laid for the cultivation of a new variety of low-chloride-content tobacco.
SEQUENCE LISTING
<110> Zhengzhou tobacco institute of China tobacco general Co
<120> a tobacco slow anion channel protein NtSLAH1 and application thereof
<130> none
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 1107
<212> DNA
<213> Nicotiana tabacum
<400> 1
atgggggaag aagtttttga atcaacaatc aaagtcacaa taagtgatga tactatgaaa 60
tcaaatgttg ccaagaaatc atcttctgct tctcttttga ctaaactaca tgcaggctat 120
ttcagaataa gcctatcttt aggtggtcaa gccttgttat ggaaagttct aattgaacat 180
ttagataaat cacaaactct tcaccactta tttcatactc tcccttcaac tactttcctc 240
ttactatggt ggatttccct ttgtaccctt ctcatcctct cttttcttta cattttaagg 300
tgcatttttc acttctcatt agttaaatca gagtttttac atcctgttgg tgtaaactat 360
ctctttgctc cttggatttc ttggctttta ttactccaat cagcaccatt tagtattcca 420
catgttggtt cttgccaagt tttttggtgg attttcgtcg ttccggttgg gattcttgat 480
gtgaaaatat atggacaatg gtttactact gagaaaaggt ttttatcaat ggttgcaaat 540
ccaactagcc aactttctgt gttgggaaat ttgactggtg cttgggttgc aagtaaaaag 600
gaatggaaag agagtgctgt ttgtatattt acattagggt taacacatta tttggtagtg 660
tttattacac tttatcaaag attatctggt agtaataacc tacctgctat gcttagacct 720
tctttctttt tgtttgtggc tgctcctagt atggctagct tagcttgggc ttctatttct 780
gggaattttg atatgtcatg cagaatgctc ttttttctct cactatttct cttcacttct 840
ttggtttgta ggccagcact attcaagaaa tcaatgagaa agttcaatgt tgcatggtgg 900
gcttactcat ttcctctcac attcctagcc ttagcctctg cacaatatgc acatcaagtg 960
aaaggtcctg tttctgctgg acttatgctg cttctctcag ccctttcagt tcttgttttt 1020
gttggtttga cagtttccac tgctctcaat cttgacatgc ttttggctga taatgatcgc 1080
tatttaaact tcactaaacg taattag 1107
<210> 2
<211> 368
<212> PRT
<213> Nicotiana tabacum
<400> 2
Met Gly Glu Glu Val Phe Glu Ser Thr Ile Lys Val Thr Ile Ser Asp
1 5 10 15
Asp Thr Met Lys Leu Asp Val Ala Lys Lys Pro Ser Ser Ala Ser Leu
20 25 30
Leu Thr Lys Leu His Ala Gly Tyr Phe Arg Ile Ser Leu Ser Leu Gly
35 40 45
Gly Gln Ala Leu Leu Trp Lys Val Leu Ile Glu His Leu Asp Lys Ser
50 55 60
Lys Thr Leu His His Leu Phe His Thr Leu Pro Ser Thr Thr Phe Leu
65 70 75 80
Leu Leu Trp Trp Ile Ser Leu Cys Thr Leu Ile Ile Leu Ser Phe Leu
85 90 95
Tyr Ile Leu Arg Cys Ile Phe His Phe Ser Leu Val Lys Ser Glu Phe
100 105 110
Leu His Pro Val Gly Val Asn Tyr Leu Phe Ala Pro Trp Ile Ser Trp
115 120 125
Leu Leu Leu Leu Gln Ser Ala Pro Phe Ser Ile Pro His Val Gly Ser
130 135 140
Cys Gln Val Phe Trp Trp Ile Phe Val Ile Pro Val Gly Ile Leu Asp
145 150 155 160
Val Lys Ile Tyr Gly Gln Trp Phe Thr Thr Glu Lys Arg Phe Leu Ser
165 170 175
Met Val Ala Asn Pro Thr Ser Gln Leu Ser Val Leu Gly Asn Leu Thr
180 185 190
Gly Ala Trp Ile Ala Ser Lys Lys Glu Trp Lys Glu Ser Ala Val Cys
195 200 205
Ile Phe Thr Leu Gly Leu Thr His Tyr Leu Val Val Phe Ile Thr Leu
210 215 220
Tyr Gln Arg Leu Ser Gly Ser Asn Ser Leu Pro Ala Met Leu Arg Pro
225 230 235 240
Ser Phe Phe Leu Phe Val Ala Ala Pro Ser Met Ala Ser Leu Ala Trp
245 250 255
Ala Ser Ile Ser Gly Asp Phe Asp Met Ser Cys Arg Met Leu Phe Phe
260 265 270
Leu Ser Leu Phe Leu Phe Thr Ser Leu Val Cys Arg Pro Ala Ile Phe
275 280 285
Lys Lys Ser Met Lys Lys Phe Asn Val Ala Trp Trp Ser Tyr Ser Phe
290 295 300
Pro Leu Thr Phe Leu Ala Leu Ala Ser Val Gln Tyr Ala His Gln Val
305 310 315 320
Lys Ser Pro Val Ser Ala Gly Leu Met Leu Leu Leu Ser Ala Leu Ser
325 330 335
Val Leu Val Phe Val Gly Leu Thr Val Ser Thr Ala Leu Asn Leu Asp
340 345 350
Met Leu Leu Ala Asp Asn Asp Arg Tyr Leu Asn Phe Thr Lys Arg Asn
355 360 365

Claims (2)

1. Coding gene of tobacco slow anion channel protein NtSLAH1NtSLAH1The application of the gene in tobacco is characterized in that the tobacco slow anion channel protein NtSLAH1 is used for transporting chloride ions; after the gene is silenced, the content of chloride ions in the tobacco leaves is obviously reduced;
the above-mentionedNtSLAH1The base sequence of the gene is shown in SEQ ID NO. 1.
2. The gene encoding the tobacco slow anion channel protein NtSLAH1 according to claim 1NtSLAH1The application of the gene in tobacco is characterized in that transient silencing VIGS vector TRV-NtSLAH1(ii) a The transient silencing VIGS vector TRV-NtSLAH1The method is specifically constructed and obtained by the following method:
the coding gene of the tobacco slow anion channel protein NtSLAH1 by using a virus-induced gene silencing technologyNtSLAH1The specific nucleotide fragment of the gene is used as a guide sequence, the specific nucleotide fragment is connected to a transient expression vector TRV, and after transformation, the transient silencing VIGS vector is further constructed and obtained through screening and identification: TRV-NtSLAH1；
The above-mentionedNtSLAH1The gene, wherein the 270 th to 615 th nucleotides are specific nucleic acid fragments.
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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
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
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CN119193674A (en) * 2024-10-22 2024-12-27 中国农业科学院烟草研究所(中国烟草总公司青州烟草研究所) Tobacco CBL family gene NtCBL10, bio-based materials containing same and applications

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