CN109354612A

CN109354612A - Tobacco AKT2/3 gene and application

Info

Publication number: CN109354612A
Application number: CN201811339759.7A
Authority: CN
Inventors: 余婧; 任学良; 李立芹; 鲁黎明; 张洁; 王仁刚; 郭玉双
Original assignee: Guizhou Institute of Tobacco Science
Current assignee: Guizhou Institute of Tobacco Science
Priority date: 2018-11-12
Filing date: 2018-11-12
Publication date: 2019-02-19
Anticipated expiration: 2038-11-12
Also published as: CN109354612B

Abstract

The present invention relates to tobacco AKT2/3 gene and applications.The sequence of the tobacco AKT2/3 gene and its coding protein is respectively as shown in SEQ ID NO:1 and 2.The clone from tobacco obtains AKT2/3 gene and demonstrates the biological function of the gene by yeast function complementation experiment the present invention for the first time, and tobacco AKT2/3 gene has the function of promoting Potassium Absorption and transhipment.

Description

Tobacco AKT2/3 gene and application thereof

Technical Field

The invention relates to the technical field of genetic engineering, in particular to a tobacco AKT2/3 gene and application thereof.

Background

Potassium ion channels are ion channels that allow potassium ions to specifically permeate the plasma membrane, while blocking the permeation of other ions, particularly sodium ions. These channels are generally composed of two parts: one part is a channel region which is selected and allows potassium ions to pass through and blocks sodium ions; the other part is a gated switch, switching channels according to signals in the environment.

The prior art studies on potassium channel genes are widespread in model plant Arabidopsis thaliana, for example, the plant Shaker ion channel, which is structurally composed of 4 subunits arranged around a central domain. The hydrophobic region of each subunit consists of 6 transmembrane regions (Trans-membrane segments TMS), of which the 4 th is a repeat of the basic residue, functionally a voltage sensing region. The highly conserved membrane loop (membrane loop) portion between the 5 th and 6 th TMS, called the P domain, constitutes a partially selective filtration region of the ion-mediated center. An important feature of the Shaker channels is that they form heterotetrameric structures, allowing plants to regulate K in different cells⁺Transport activity, this regulation being independent in each organ or tissue and related to environmental conditions.

Tobacco is a crop with large potassium consumption, the potassium content of tobacco leaves is an important index for measuring the quality of the tobacco leaves, at present, researches on potassium ion channels in the tobacco are few, and the function of a tobacco Shaker is unknown.

Disclosure of Invention

The invention aims to provide a tobacco AKT2/3 gene and a protein coded by the gene.

Another purpose of the invention is to provide application of the tobacco AKT2/3 gene.

In order to achieve the object of the present invention, the present invention provides a tobacco AKT2/3 gene encoding the following protein (a) or (b):

(a) a protein consisting of an amino acid sequence shown as SEQ ID NO. 2;

(b) 2, protein which is derived from (a) and has the same function by substituting, deleting or adding one or more amino acids in the sequence shown in SEQ ID NO. 2.

The nucleotide sequence of the tobacco AKT2/3 gene is shown as SEQ ID NO. 1, and the full length of the gene is 2355 bp. The invention adopts the following method to clone and obtain the tobacco AKT2/3 gene:

① before the AKT2/3 gene PCR amplification, extracting the total RNA of the tobacco cells, and reversely transcribing the extracted total RNA into cDNA. in the invention, the total RNA of the tobacco cells is extracted by adopting the technical scheme of extracting the total RNA of the cells commonly used in the field, and the Trizol method can be specifically adopted in the embodiment of the invention.

② and reverse transcribing the total RNA of the tobacco cells to synthesize the cDNA, wherein the cDNA is synthesized by a conventional cDNA synthesis method in the field without other special requirements, and the cDNA synthesis is completed by a cDNA synthesis kit of TaKaRa company in the embodiment of the invention.

③ after obtaining cDNA, the AKT2/3 gene PCR amplification is carried out to obtain the target fragment, in the invention, the AKT2/3 gene PCR amplification is carried outThe enhanced system is preferably 20 μ L system, including Premix ExTaq 10 μ L, forward primer 0.5 μ L at 10 μ M, reverse primer 0.5 μ L at 10 μ M, tobacco cell cDNA 1 μ L, ddH₂O8. mu.L. In the present invention, the reaction procedure for the PCR amplification of AKT2/3 gene is preferably: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s; annealing at 55 ℃ for 30 s; extending for 2min at 72 ℃; 35 cycles.

④ after the AKT2/3 gene PCR amplification to get the target fragment, sequencing the target fragment to get AKT2/3 gene the invention preferably purifies the target fragment after the PCR amplification, the invention does not limit the purification method, and it can use the DNA purification kit well known by the technicians in this field.

⑤ after purification, the purified target fragment is preferably introduced into Escherichia coli DH5 α competent cells to perform colony PCR and sequence after verifying positive clone, the invention preferably adopts a colony PCR method to verify positive clone after obtaining positive clone, in the invention, the nucleotide sequence of the forward primer of the colony PCR is 5'-ATGCAAATGAGCCATTCATC-3' (SEQ ID NO:3), the nucleotide sequence of the reverse primer is 5'-CTACAAGCAATTTGGATCT-3' (SEQ ID NO:4), the system of the colony PCR is 10 muL, and the system comprises Premix ExTaq5 muL, 10 muM forward primer 0.5 muL, 10 muM reverse primer 0.5 muL, ddH₂The method for introducing the purified target fragment into the escherichia coli DH5 α competent cells is a conventional method for transforming the escherichia coli competent cells in the field, and specifically comprises the steps of connecting the target fragment and a pMD19-T vector at 16 ℃ for 10-14 hours to obtain a connection product, transforming the connection product into the escherichia coli DH5 α competent cells to obtain transformed escherichia coli DH5 α, and inoculating the transformed escherichia coli DH5 α onto an LB plate coated with ampicillin to perform screening culture to obtain positive clones.

⑥ after positive clones are verified by colony PCR, preferably, 2-4 independent positive clones are randomly selected from the verified positive clones for sequencing to obtain the sequence of the tobacco AKT2/3 gene.

The invention also provides a biological material containing the tobacco AKT2/3 gene, wherein the biological material is an expression cassette, an expression vector, a cloning vector, an engineering bacterium or a transgenic cell line.

The invention also provides application of the tobacco AKT2/3 gene or a biological material containing the gene in promoting absorption and transportation of plant or microorganism potassium ions.

The plants of the invention include but are not limited to tobacco and arabidopsis thaliana. Such microorganisms include, but are not limited to, yeast.

The invention also provides application of the tobacco AKT2/3 gene or biological material containing the gene in preparation of transgenic plants.

The invention also provides application of the tobacco AKT2/3 gene or biological material containing the gene in plant breeding. The breeding aim is to promote the absorption and the transportation of plant potassium ions.

Preferably, the tobacco AKT2/3 gene is transferred into a tobacco plant, so that the tobacco AKT2/3 gene is over-expressed to improve the content of potassium ions in tobacco leaves of the tobacco plant. More preferably, the tobacco AKT2/3 gene is transferred into tobacco plants by adopting an agrobacterium-mediated method to obtain transgenic plants with over-expressed AKT2/3 gene.

The invention also provides a specific PCR primer pair for amplifying the tobacco AKT2/3 gene, wherein the nucleotide sequence of the primer pair is shown as SEQ ID NO. 3-4. The primer pair is designed by using software primer5 and using NCBI Reference Sequence LOC107761957 as a Reference Sequence.

The invention also provides a method for promoting the absorption and the transportation of plant potassium ions, which comprises the following steps:

1) allowing a plant to comprise said tobacco AKT2/3 gene; or,

2) allowing the plant to overexpress the tobacco AKT2/3 gene.

Such methods include, but are not limited to, transgenics, crosses, backcrosses, selfs, or asexual propagation.

The invention clones AKT2/3 gene from tobacco for the first time, verifies the biological function of the gene through yeast function complementation experiment, and the recombinant yeast after the tobacco AKT2/3 gene is transferred into potassium absorption defective yeast mutant R5421 has potassium ion absorption and transfer functions. Therefore, the tobacco AKT2/3 gene provided by the invention has the function of promoting potassium ion absorption and transportation.

Drawings

FIG. 1 shows the results of the yeast function complementation test in example 2 of the present invention. Wherein, A: the concentration of potassium ions in the medium was 20uM, B: the potassium ion concentration in the medium was 2 mM. In the figure, 1 is recombinant yeast transferred with tobacco AKT2/3 gene, 2 is negative control group (transferred with empty vector), 3 is recombinant yeast of positive control group (transferred with Arabidopsis AtAKT1 gene); the growth results of the strain stock solution, the 10-time diluent, the 100-time diluent and the 1000-time diluent on the culture medium are sequentially shown from left to right.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular Cloning handbook, Sambrook et al (Sambrook J & Russell DW, Molecular Cloning: a Laboratory Manual,2001), or the conditions as recommended by the manufacturer's instructions.

Example 1 cloning of the tobacco AKT2/3 Gene

Taking 0.5g of fresh tobacco leaf (tobacco variety K326), extracting total RNA of tobacco cell by Trizol method, synthesizing cDNA by cDNA synthesis kit of TaKaRa company, and further adopting Primer5.0 softThe primer is designed and obtained through artificial optimization, the primer comprises a forward primer and a reverse primer, and the nucleotide sequence of the forward primer is as follows: 5'-ATGCAAATGAGCCATTCATC-3', respectively; the nucleotide sequence of the reverse primer is 5'-CTACAAGCAATTTGGATCT-3', synthesized cDNA is taken as a template to carry out PCR amplification, the PCR amplification system is a 20 mu L system which comprises 10 mu L of Premix ExTaq, 0.5 mu L of 10 mu M forward primer, 0.5 mu L of 10 mu M reverse primer, 1 mu L of tobacco cell cDNA and ddH₂O8 mu L; the reaction procedure of the PCR amplification is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30 s; annealing at 55 ℃ for 30 s; extending for 2min at 72 ℃; 35 cycles.

After PCR amplification is finished, a DNA purification kit is used for purifying a target fragment, the purified target fragment is connected with a pMD19-T vector for 12 hours at the temperature of 16 ℃ to obtain a connection product, the obtained connection product is converted into escherichia coli DH5 α competent cells to obtain converted escherichia coli DH5 α, the converted escherichia coli DH5 α is inoculated on an LB plate coated with ampicillin for screening and culture to obtain positive clones, after the positive clones are obtained, a colony PCR method is adopted to verify the positive clones, the forward primer of the colony PCR is 5'-ATGCAAATGAGCCATTCATC-3', the reverse primer is 5'-CTACAAGCAATTTGGATCT-3', the colony PCR system is 10 mu L and comprises Premix ExTaq5 mu L, the forward primer of 10 mu M is 0.5 mu L, the reverse primer of 10 mu M is 0.5 mu L, and ddH is 10 mu L₂O4. mu.L. Then randomly selecting 3 independent positive clones from the verified positive clones, sending the positive clones to a biotechnology company for sequencing, and obtaining the sequence of the tobacco AKT2/3 gene as shown in SEQ ID NO:1 through sequencing.

Example 2 biological functional analysis of the tobacco AKT2/3 Gene

1. Purpose of experiment

The biological function of the tobacco AKT2/3 gene is verified by a yeast function complementation experiment.

2. Experimental methods

The potassium absorption-deficient yeast mutant strain R5421 was used as a recipient bacterium. Strain R5421 can be found in Maathuis F J Manual, Sanders D1996 mechanics of potassium adsorption by highher plants Physiol.plant.96, 158-168.

The T-vector connected with the tobacco AKT2/3 Gene in example 1 and an Expression vector P416 (yeast free shuttle Expression vector, TEF constitutive promoter, CYC1 terminator, CEN6 ARSH4 replication origin, URA3 in yeast, Amp. vector P416 in Escherichia coli, Functional Expression of a omega-3 Fatty acid desaturase Gene from Glycine max in Saccharomyces cerevisiae) are subjected to double enzyme digestion (enzyme digestion sites are Xba I and Xho I) respectively, the target Gene and the Expression vector P416 are recovered and then connected by ligase, the connected recombinant yeast Expression vector is transferred into competent cells of Escherichia coli DH5 α, and PCR amplification and enzyme digestion are carried out on the transformed Escherichia coli single colony to verify whether the construction is successful.

The specific steps of transferring the successfully constructed recombinant yeast expression vector into the yeast R5421 are as follows: taking the preserved R5421 yeast by an inoculating ring, streaking on a solid culture medium YPDA, and culturing at 28 ℃ for 12 h; picking a single colony of the R5421 yeast in an Ep tube, adding 1mL of YPDA culture solution, and vortexing; transferring all the above bacterial liquid into a triangular flask containing YPDA culture solution, and shaking at 30 deg.C and 250rpm to OD₆₀₀1.2, 16 h; transferring according to the volume ratio of 1:10, and shaking to OD₆₀₀1.0-1.2; centrifuging at 28 deg.C and 1000rpm for 5min, and resuspending with 1/2 volume of sterilized ultrapure water; centrifuging at 28 deg.C and 1000rpm for 5min for collecting bacteria, and sucking off supernatant; the following ingredients (per 5mL of original bacterial liquid) were added in sequence:

vortex for 1min to make the transformation system completely mixed; placing in water bath at 30 deg.C, and incubating for 30 min; placing in 42 deg.C water bath, thermally shocking for 28min, and cooling on ice for 10 min; centrifuging at 7000rpm for 15s, and discarding the supernatant; gently resuspend the pellet with 1mL of sterile water; spreading 200. mu.L of the transformation mixture on an auxotrophic plate; cultured at 30 ℃ for 3 days. Yeast plasmids were extracted and transformation results were identified.

Selecting identified yeast single colony, streaking on auxotrophic plate, and culturing at 30 deg.C for 3 days; dipping a small amount of thallus on an auxotrophic flat plate by using a toothpick, and culturing in 2mL of auxotrophic liquid (8 g of Ura Minus Media, 20g of glucose and 7.5g of potassium chloride, adjusting the pH value to 5.8 by using NaOH, and fixing the volume to 1000mL) for 12 h; centrifuging at 8000rpm for 1min, and collecting thallus; discarding the supernatant, suspending the thallus with 1mL of double distilled water, and centrifuging at 8000rpm for 1 min; discarding the supernatant, resuspending with 1ml of double distilled water, and adjusting OD₆₀₀Is 0.8; the undiluted bacterial solution and 10-fold and 100-fold diluted bacterial solutions were cultured in 5uL of 0 and 2mM potassium ion medium at 30 ℃ for 3 days, and the results were observed.

3. Results of the experiment

As shown in FIG. 1, the yeast of the negative control group (transferred into P416 empty vector) hardly grew, and both the recombinant yeast of the tobacco AKT2/3 gene and the recombinant yeast of the positive control group (transferred into Arabidopsis thaliana AtAKT1 gene) could grow on a 2mM medium (AP medium (1L): 546. mu.L phosphate, 1.742g L-arginine, 1mL 1000 Xvitamin solution, 1mL 1000 Xmicroelement solution, 0.77g uracil, 10mL 100 XUra, 20g glucose, 15g agar powder) with potassium ion concentration. With the increase of the dilution factor, the recombinant yeast transferred into the tobacco AKT2/3 gene and the recombinant yeast of the positive control group can still grow. The results prove that the tobacco AKT2/3 gene has potassium absorption and transport functions.

Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.

Sequence listing

<110> Guizhou province tobacco science research institute

<120> tobacco AKT2/3 gene and application

<130>KHP171117871.4

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>2355

<212>DNA

<213> tobacco (Nicotiana tabacum)

<400>1

atgcaaatga gccattcatc ttatagtgtg tacaatagct ctacaaaaaa catgataatt 60

gaagatagcc aaattaaaga ccaacatgtg caagataata gccatgggag tagcaataat 120

tcagatgagc tcagttttcg aaacctgtca aagctcattc tacctcctct tggttccaat 180

ggttacaacc agaatcaaac tcaacagaaa ggcaagacca tcacccctat ggattcaaga 240

tacaggtgtt gggagacgct aatggtaata atggtggcgt attctgcatg ggtatgtcca 300

tttgagatag cattcatgca ctctaaccca aatagagtgc tatacatcgc agacaacgtt 360

gttgatctct tctttgcgat tgatatcatc ttgactttct ttgttgccta tattgatact 420

actactcagc ttctcgtacg tgacaggaaa agaattgcta caagacttga aaaggacatg 480

agattcagct atttttgggt cagatgtgcc aggcttctat ttgtaacact attgacagtg 540

cactgtgctg gatgcctcta ctatttgcta gctgatagat atccacacca aggagatact 600

tggttaggag ctatgaatcc aaattacaag gagacaagtc ttcttattag gtatattgca 660

gctttatatt ggtccattac taccatgaca acagttggct atggtgatct ccatgctgtc 720

aacactttgg aaatggtctt catcattttc tatatgctct tcaatcttgg tctcactgct 780

tatattattg gtaatatgac caatttagtt gtcgaaggaa ctcgtcgcac aatggaattc 840

agaaatagca tcgaagcagc atcaaatttc gtgtgtcgaa acaggttgcc tccaagatta 900

aaagagcaaa tattggctta catgtgttta agattcaggg cagagagcct aaaccaacag 960

caattgattg aacaacttcc caagacaatc tgcaaaagca ttaggcacca tttgtttttg 1020

ccaacagtag agaaggttta tcttttcaag ggtgtctcaa gggaaattct gttacttttg 1080

gttgcagata cgaaggctga gtacataccc ccaagagagg atgtaataat gcagaacgaa 1140

tcgccagatg aggtttacat catagtgtca ggggaggtgg aaatgattga gtctgagatg 1200

gaaaatgagc aggttgtttg gacattcaaa tctggagata tgttaggaga agtcggggca 1260

ttttgccgta ggcctcagag ctacacgtat cgaaccaaga cactttcaca actcctgaag 1320

ataagaacaa cttctttgat tgaagcaatg aaaaccagac aagaagataa tatcataatg 1380

atcaagaatt tccttcagca tcacaagaag ctcagggatt taaagcttgg agatttattt 1440

catgcagttg gggaagaaaa tggtgatcca aacatgtctg ttaatttgct aactgttgct 1500

agtacaggta atgctacttt tcttgaggaa cttctcaagg caagatttga tcctgatatc 1560

ggagatgccc aaggaagaac cccactgcac atagcagcat cgaaagggca cgaagagtgt 1620

gtgatggtac ttcttagaca tggatgtaac atatacctcc cagatgtaaa tggtaacacc 1680

gcgttgtggg aagctatagc agcaaaacaa cattcaacat ttcggatatt ataccattgg 1740

gcttctgtct ccgatcctta tgttgctggt gaactcttat gcaccgcagc taagagaaat 1800

gacataacag tgatgaaaga actcctaaaa catggattaa cagttgactc aaaagatcgc 1860

cacggatcaa ctgctattga tgttgcattg ggagaaaatc atgaggacat ggtaaagcta 1920

cttctaatga atggagccga gataaatgat aaatttaagc acaagctttc ttcgatgaac 1980

ctaagagaga ttctacaaaa acgagaagta gggcacaggg ttatagttcc cgacacaatg 2040

gatgaagttg ctcaaaagtg gcgcgaacaa gagcaaaagt acaactcggg aagtactagg 2100

gatcaatcgt cttttagagt tagcatatac aaaggccatc cggtgatcag gaaaagaact 2160

cattgctgtg aaccagggaa attaataatg cttccaaatt cacttgcaga gctcaagatc 2220

attgcaggtc agaaatttgg atttgatgca acaaaagcat tggtgacaga tcaagaagga 2280

tcagaaattg actctattga agtgataaga gataatgata agctgtatat agttgaagat 2340

ccaaattgct tgtag 2355

<210>2

<211>784

<212>PRT

<213> tobacco (Nicotiana tabacum)

<400>2

Met Gln Met Ser His Ser Ser Tyr Ser Val Tyr Asn Ser Ser Thr Lys

1 5 10 15

Asn Met Ile Ile Glu Asp Ser Gln Ile Lys Asp Gln His Val Gln Asp

20 25 30

Asn Ser His Gly Ser Ser Asn Asn Ser Asp Glu Leu Ser Phe Arg Asn

35 40 45

Leu Ser Lys Leu Ile Leu Pro Pro Leu Gly Ser Asn Gly Tyr Asn Gln

50 55 60

Asn Gln Thr Gln Gln Lys Gly Lys Thr Ile Thr Pro Met Asp Ser Arg

65 70 75 80

Tyr Arg Cys Trp Glu Thr Leu Met Val Ile Met Val Ala Tyr Ser Ala

85 90 95

Trp Val Cys Pro Phe Glu Ile Ala Phe Met His Ser Asn Pro Asn Arg

100 105 110

Val Leu Tyr Ile Ala Asp Asn Val Val Asp Leu Phe Phe Ala Ile Asp

115 120125

Ile Ile Leu Thr Phe Phe Val Ala Tyr Ile Asp Thr Thr Thr Gln Leu

130 135 140

Leu Val Arg Asp Arg Lys Arg Ile Ala Thr Arg Leu Glu Lys Asp Met

145 150 155 160

Arg Phe Ser Tyr Phe Trp Val Arg Cys Ala Arg Leu Leu Phe Val Thr

165 170 175

Leu Leu Thr Val His Cys Ala Gly Cys Leu Tyr Tyr Leu Leu Ala Asp

180 185 190

Arg Tyr Pro His Gln Gly Asp Thr Trp Leu Gly Ala Met Asn Pro Asn

195 200 205

Tyr Lys Glu Thr Ser Leu Leu Ile Arg Tyr Ile Ala Ala Leu Tyr Trp

210 215 220

Ser Ile Thr Thr Met Thr Thr Val Gly Tyr Gly Asp Leu His Ala Val

225 230 235 240

Asn Thr Leu Glu Met Val Phe Ile Ile Phe Tyr Met Leu Phe Asn Leu

245 250 255

Gly Leu Thr Ala Tyr Ile Ile Gly Asn Met Thr Asn Leu Val Val Glu

260 265 270

Gly Thr Arg Arg Thr Met Glu Phe Arg Asn Ser Ile Glu Ala Ala Ser

275 280 285

Asn Phe Val Cys Arg Asn Arg Leu Pro Pro Arg Leu Lys Glu Gln Ile

290 295 300

Leu Ala Tyr Met Cys Leu Arg Phe Arg Ala Glu Ser Leu Asn Gln Gln

305 310 315 320

Gln Leu Ile Glu Gln Leu Pro Lys Thr Ile Cys Lys Ser Ile Arg His

325 330 335

His Leu Phe Leu Pro Thr Val Glu Lys Val Tyr Leu Phe Lys Gly Val

340 345 350

Ser Arg Glu Ile Leu Leu Leu Leu Val Ala Asp Thr Lys Ala Glu Tyr

355 360 365

Ile Pro Pro Arg Glu Asp Val Ile Met Gln Asn Glu Ser Pro Asp Glu

370 375 380

Val Tyr Ile Ile Val Ser Gly Glu Val Glu Met Ile Glu Ser Glu Met

385 390 395 400

Glu Asn Glu Gln Val Val Trp Thr Phe Lys Ser Gly Asp Met Leu Gly

405 410 415

Glu Val Gly Ala Phe Cys Arg Arg Pro Gln Ser Tyr Thr Tyr Arg Thr

420 425 430

Lys Thr Leu Ser Gln Leu Leu Lys Ile Arg Thr Thr Ser Leu Ile Glu

435 440 445

Ala Met Lys Thr Arg Gln Glu Asp Asn Ile Ile Met Ile Lys Asn Phe

450 455 460

Leu Gln His His Lys Lys Leu Arg Asp Leu Lys Leu Gly Asp Leu Phe

465 470 475 480

His Ala Val Gly Glu Glu Asn Gly Asp Pro Asn Met Ser Val Asn Leu

485 490 495

Leu Thr Val Ala Ser Thr Gly Asn Ala Thr Phe Leu Glu Glu Leu Leu

500 505 510

Lys Ala Arg Phe Asp Pro Asp Ile Gly Asp Ala Gln Gly Arg Thr Pro

515 520 525

Leu His Ile Ala Ala Ser Lys Gly His Glu Glu Cys Val Met Val Leu

530 535 540

LeuArg His Gly Cys Asn Ile Tyr Leu Pro Asp Val Asn Gly Asn Thr

545 550 555 560

Ala Leu Trp Glu Ala Ile Ala Ala Lys Gln His Ser Thr Phe Arg Ile

565 570 575

Leu Tyr His Trp Ala Ser Val Ser Asp Pro Tyr Val Ala Gly Glu Leu

580 585 590

Leu Cys Thr Ala Ala Lys Arg Asn Asp Ile Thr Val Met Lys Glu Leu

595 600 605

Leu Lys His Gly Leu Thr Val Asp Ser Lys Asp Arg His Gly Ser Thr

610 615 620

Ala Ile Asp Val Ala Leu Gly Glu Asn His Glu Asp Met Val Lys Leu

625 630 635 640

Leu Leu Met Asn Gly Ala Glu Ile Asn Asp Lys Phe Lys His Lys Leu

645 650 655

Ser Ser Met Asn Leu Arg Glu Ile Leu Gln Lys Arg Glu Val Gly His

660 665 670

Arg Val Ile Val Pro Asp Thr Met Asp Glu Val Ala Gln Lys Trp Arg

675 680685

Glu Gln Glu Gln Lys Tyr Asn Ser Gly Ser Thr Arg Asp Gln Ser Ser

690 695 700

Phe Arg Val Ser Ile Tyr Lys Gly His Pro Val Ile Arg Lys Arg Thr

705 710 715 720

His Cys Cys Glu Pro Gly Lys Leu Ile Met Leu Pro Asn Ser Leu Ala

725 730 735

Glu Leu Lys Ile Ile Ala Gly Gln Lys Phe Gly Phe Asp Ala Thr Lys

740 745 750

Ala Leu Val Thr Asp Gln Glu Gly Ser Glu Ile Asp Ser Ile Glu Val

755 760 765

Ile Arg Asp Asn Asp Lys Leu Tyr Ile Val Glu Asp Pro Asn Cys Leu

770 775 780

<210>3

<211>20

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>3

atgcaaatga gccattcatc 20

<210>4

<211>19

<212>DNA

<213> Artificial Sequence (Artificial Sequence)

<400>4

ctacaagcaa tttggatct 19

Claims

1. A tobacco AKT2/3 gene, which is a gene encoding the following protein (a) or (b):

(a) a protein consisting of an amino acid sequence shown as SEQ ID NO. 2;

2. The gene of claim 1, wherein the nucleotide sequence is represented by SEQ ID NO 1.

3. Biological material comprising the gene of claim 1 or 2, said biological material being an expression cassette, an expression vector, a cloning vector or an engineered bacterium.

4. Use of the gene of claim 1 or 2 or the biomaterial of claim 3 for promoting plant or microbial potassium ion uptake and transport.

5. The use of claim 4, wherein the plant comprises tobacco, Arabidopsis, and the microorganism comprises yeast.

6. Use of the gene according to claim 1 or 2 or the biological material according to claim 3 for the preparation of transgenic plants.

7. Use of the gene of claim 1 or 2 or the biomaterial of claim 3 in plant breeding.

8. The use of claim 7, wherein the breeding is aimed at promoting plant potassium ion uptake and transport.

9. A method for promoting plant potassium ion absorption and transport, which is characterized by comprising the following steps:

1) allowing a plant to comprise the gene of claim 1 or 2; or,

2) overexpressing in a plant a gene according to claim 1 or 2;

wherein the plant comprises tobacco and arabidopsis thaliana.

10. The method of claim 9, wherein the method comprises transgenesis, crossing, backcrossing, selfing, or asexual propagation.