CN109438564B

CN109438564B - Tobacco KUP6 protein and coding gene and application thereof

Info

Publication number: CN109438564B
Application number: CN201811367081.3A
Authority: CN
Inventors: 王自力; 任学良; 李立芹; 鲁黎明; 王仁刚; 张洁; 郭玉双
Original assignee: Guizhou Institute of Tobacco Science
Current assignee: Guizhou Institute of Tobacco Science
Priority date: 2018-11-16
Filing date: 2018-11-16
Publication date: 2021-08-31
Anticipated expiration: 2038-11-16
Also published as: CN109438564A

Abstract

The invention relates to the field of genetic engineering, and particularly discloses a tobacco KUP6 protein, and a coding gene and application thereof. The invention provides a KUP6 gene separated from tobacco for the first time, wherein the nucleotide sequence of the KUP6 gene is shown as SEQ ID NO.2, the amino acid sequence of the coded protein KUP6 is shown as SEQ ID NO.1, and through functional verification, after the KUP6 gene provided by the invention is transferred into a potassium absorption defective yeast mutant strain R5421, a recombinant yeast expressing the KUP6 gene has potassium ion absorption and transfer functions. The KUP6 gene provided by the invention has the function of promoting potassium ion absorption and transport.

Description

Tobacco KUP6 protein and coding gene and application thereof

Technical Field

The invention relates to the field of genetic engineering, in particular to a potassium transport protein KUP6 from tobacco, and a coding gene and application thereof.

Background

The potassium transporter is a carrier protein capable of transporting potassium ions into cells when the concentration of external potassium ions is extremely low. Potassium transporters are generally transmembrane proteins, and transport of potassium ions across the membrane is accomplished by conformational changes, an active transport process requiring energy from ATP, usually K⁺/H⁺Or K⁺/Na⁺And (5) carrying out coordinated transportation.

The research on potassium transporter genes in the prior art is relatively extensive in model plant Arabidopsis, for example, the research shows that the survival rate of an Arabidopsis mutant KUP6 is reduced compared with that of a wild plant under drought stress, and 35S shows that the drought tolerance of KUP6 overexpression transgenic plants is obviously enhanced (Shabala et al, 2007); and mutant seedlings of KUP7 gene show a low-potassium sensitive phenotype with etiolated leaves under low potassium stress, the content of potassium ions at roots of the mutant seedlings is obviously reduced, and the loss of the function of KUP7 gene reduces the potassium absorption capacity of arabidopsis seedlings under low potassium conditions and the concentration of potassium ions in xylem sap (Min et al, 2016).

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 transporter genes in the tobacco are less, and the function of KUP of the tobacco is unknown.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a tobacco KUP6 protein, a coding gene and application thereof.

In order to realize the purpose of the invention, the technical scheme of the invention is as follows:

the invention firstly provides a potassium transport protein which is obtained from tobacco and is named as KUP6 protein and is (a) or (b)

(a) The amino acid sequence is shown as SEQ ID NO. 1;

(b) and (b) a protein which is derived from the protein (a) and has the same function as the protein (a) through substitution and/or deletion and/or addition of one or more amino acid residues in the sequence shown in SEQ ID NO. 1.

The invention provides a gene for coding the tobacco KUP6 protein, wherein the gene is a DNA molecule of the following 1) or 2) or 3):

1) the coding region is a DNA molecule shown as SEQ ID NO. 2;

2) a DNA molecule which is hybridized with the DNA sequence defined in 1) under strict conditions and codes a protein related to the regulation and control of plant potassium ion absorption and transport;

3) DNA molecules which have more than 90 percent of homology with the DNA sequences limited by 1) or 2) and encode proteins related to plant potassium ion absorption and transport regulation.

The research shows that the KUP6 gene plays a significant role in promoting potassium ion absorption and transport.

Further, the KUP6 gene of the invention is prepared by the following steps:

designing PCR amplification primers, wherein the PCR amplification primers comprise a forward primer and a reverse primer: the nucleotide sequence of the forward primer is: 5'-ATGGCGAGCTCAGATAGTGA-3', the nucleotide sequence of the reverse primer is 5'-CTATAGTTCATAAGTCATGC-3'; (2) extracting total RNA of the tobacco cells, synthesizing cDNA of the tobacco cells, carrying out PCR amplification of KUP6 gene by taking the cDNA of the tobacco cells as a template to obtain a target segment, and sequencing.

Preferably, the PCR amplification system is a 20 μ L system, including Premix ExTaq 10 μ L, forward primer 0.5 μ L of 10 μ M, reverse primer 0.5 μ L of 10 μ M, tobacco cell cDNA 1 μ L, ddH₂O 8μL。

Preferably, 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.

Preferably, the target fragment is introduced into escherichia coli DH5 alpha competent cells for colony PCR verification before sequencing, and sequencing is performed after positive clones are verified.

Preferably, the nucleotide sequence of the forward primer used for colony PCR verification is as follows: the nucleotide sequence of the forward primer is: 5'-ATGGCGAGCTCAGATAGTGA-3', the nucleotide sequence of the reverse primer used was verified to be 5'-CTATAGTTCATAAGTCATGC-3' by colony PCR.

Preferably, the colony PCR verification system is 10 μ L, including Premix ExTaq5 μ L, 10 μ M forward primer 0.5 μ L, 10 μ M reverse primer 0.5 μ L, ddH₂O 4μL。

The invention provides a biological material containing the gene for coding the KUP6 protein of the tobacco, and the biological material is a recombinant expression vector, an expression cassette, a transgenic cell line or a recombinant bacterium.

The recombinant expression vector can be constructed by using the existing plant expression vector. The recombinant expression vector can be, for example, a binary Agrobacterium vector, a vector useful for microprojectile bombardment of plants, and the like. When the KUP6 gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters can be added in front of the transcription initiation nucleotide, and can be used alone or combined with other plant promoters; in addition, when constructing a recombinant expression vector using KUP6 gene, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene.

The recombinant expression vector carrying the KUP6 gene can be transformed into plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium mediation and the like.

The invention provides an application of the tobacco KUP6 protein or the coding gene thereof or the biological material containing the gene in promoting the absorption and the transportation of potassium ions of plants or microorganisms.

The plant comprises tobacco and arabidopsis thaliana.

The microorganism includes yeast.

For example, in the embodiment of the present invention, after the KUP6 gene is transferred into the potassium absorption deficient yeast mutant R5421, the recombinant yeast expressing the KUP6 gene has potassium ion absorption and transport functions again; after the KUP6 gene in the tobacco plant is subjected to overexpression, the content of potassium ions in tobacco leaves of the tobacco plant can be obviously improved.

The application can be selected as that the KUP6 gene of the tobacco is transferred into a tobacco plant, and the KUP6 gene is overexpressed to improve the content of potassium ions in tobacco leaves of the tobacco plant.

The invention provides an application of the tobacco KUP6 protein or the coding gene thereof or the biological material containing the gene in preparing transgenic plants.

The invention provides the application of the tobacco KUP6 protein or the coding gene thereof or the biological material containing the gene in plant breeding.

The breeding aim is to promote or regulate plant potassium ion absorption and transport.

The plant is tobacco.

The invention has the beneficial effects that: the invention provides a KUP6 gene separated from tobacco for the first time, wherein the nucleotide sequence of the KUP6 gene is shown as SEQ ID NO.2, and the amino acid sequence of the coding protein is shown as SEQ ID NO. 1. Through functional verification, after the KUP6 gene provided by the invention is transferred into a potassium absorption deficient yeast mutant strain R5421 (the mutant strain is described in Maathuis F J M and Sanders D1996 mechanics of potassium absorption by high plant roots. Physiol. plant.96, 158-168.), the recombinant yeast expressing the KUP6 gene has the potassium ion absorption and transport functions again. The KUP6 gene provided by the invention has the function of promoting potassium ion absorption and transport.

Drawings

FIG. 1A shows the results of the yeast complementation test in example 2 of the present invention, wherein the growth was performed on a medium having a potassium ion concentration of 20uM, and FIG. 1B shows the growth on a medium having a potassium ion concentration of 2 mM; in the figure, 1 is a negative control (transferred into an empty vector), 2 is a recombinant yeast transferred into KUP6 gene, and 3 is a positive control yeast transferred into Arabidopsis thaliana KUP gene; the growth results of the 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

Preferred embodiments of the present invention will be described in detail with reference to the following examples. It is to be understood that the following examples are given for illustrative purposes only and are not intended to limit the scope of the present invention. Various modifications and alterations of this invention will become apparent to those skilled in the art without departing from the spirit and scope of this invention.

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Example 1 acquisition of KUP6 Gene

Taking 0.5g of fresh tobacco leaves, extracting total RNA of tobacco cells by a Trizol method, synthesizing cDNA by a cDNA synthesis kit of TaKaRa company, further designing by Primer5.0 software and obtaining primers through artificial optimization, wherein the primers comprise a forward primer and a reverse primer, and the nucleotide sequence of the forward primer is as follows: 5'-ATGGCGAGCTCAGATAGTGA-3', the nucleotide sequence of the reverse primer is 5'-CTATAGTTCATAAGTCATGC-3'; . PCR amplification was performed using the synthesized cDNA as a template.

The PCR amplification system is a 20 mu L system and comprises: premix ExTaq 10. mu.L, forward primer 0.5. mu.L at 10. mu.M, reverse primer 0.5. mu.L at 10. mu.M, tobacco cell cDNA 1. mu.L, ddH₂O 8μL。

The PCR amplification reaction program is as follows: pre-denaturation at 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, extension at 72 ℃ for 2min, 35 cycles.

After PCR amplification is completed, 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 16 ℃ to obtain a connection product, the obtained connection product is transformed into escherichia coli DH5 alpha competent cells to obtain transformed escherichia coli DH5 alpha, and the transformed escherichia coli DH5 alpha is inoculated on an LB plate coated with ampicillin to be screened and cultured to obtain a positive clone. After obtaining the positive clone, verifying the positive clone by adopting a colony PCR method, wherein the nucleotide sequence of a forward primer of the colony PCR is as follows: 5'-ATGGCGAGCTCAGATAGTGA-3', the nucleotide sequence of the reverse primer is 5'-CTATAGTTCATAAGTCATGC-3'; the colony PCR system is 10 μ L, including 5 μ L of Premix ExTaq, 0.5 μ L of 10 μ M forward primer, 0.5 μ L of 10 μ M reverse primer, ddH₂O4. mu.L. Then randomly selecting 3 independent positive clones from the verified positive clones, sending the positive clones to a biotechnology company for sequencingThe sequence of the KUP6 gene obtained after sequencing is shown as SEQ ID NO.2, and the amino acid sequence is shown as SEQ ID NO. 1.

Example 2 Effect of KUP6 Gene on promoting Potassium ion absorption and transport

The construction of the recombinant yeast Expression vector was verified by performing PCR amplification and enzyme digestion on a single colony of transformed E.coli, and the constructed recombinant yeast Expression vector was successfully transferred into R5421 by performing double enzyme digestion on a T-vector to which the KUP6 Gene described in example 1 was ligated and an Expression vector P416(P416 yeast free shuttle Expression vector, TEF constitutive promoter, CYC1 terminator, CEN6ARSH4 replication origin, screening marker URA3 in yeast, screening marker Amp in E.coli, restriction sites: Sma I and Xho I), respectively, recovering the target Gene and the Expression vector P416, ligating them with ligase, transferring the ligated recombinant yeast Expression vector into competent cells of E.coli DH5 α, performing PCR amplification and enzyme digestion on the transformed E.coli single colony.

The method comprises the following specific steps: 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 bacterial liquid into a triangular flask filled with YPDA culture solution, and shaking at 30 ℃ and 250rpm until the OD600 is 1.2; switching over according to the ratio of 1:10, and shaking until OD600 is 1.0-1.2; 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; incubating in 30 deg.C water bath 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. And extracting yeast plasmids and identifying the result.

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 the auxotrophic flat plate by using a toothpick, and culturing in 2mL of auxotrophic solution 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;

discard the supernatant, resuspend with 1mL double distilled water, adjust OD₆₀₀Is 0.8; the undiluted bacterial solution and the 10-fold and 100-fold diluted bacterial solutions were cultured in 5uL of 2mM potassium ion medium at 30 ℃ for 3 days, respectively, and the results were observed.

As shown in FIGS. 1A and 1B, the yeast of the negative control group (into which P416 empty vector was transferred) hardly grew, and both the recombinant yeast of the tobacco KUP6 gene and the recombinant yeast of the positive control group (into which Arabidopsis thaliana KUP gene was transferred) grew, on a 2mM medium (AP medium (1L): 546. mu.L phosphate, 1.742g of L-arginine, 1mL of 1000X vitamin solution, 1mL of 1000X trace element solution, 0.77g of uracil, 100X Ura 10mL of glucose, 20g of agar powder, and 15g of the like) in which the potassium ion concentration was 20 uM. With the increase of dilution factor, the recombinant yeast transferred into the KUP6 gene of tobacco and the recombinant yeast of the positive control group can still grow.

The results prove that the KUP6 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 KUP6 protein and coding gene and application thereof

<130> KHP171117857.8

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 805

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Ala Ser Ser Asp Ser Glu Arg His Thr Glu Glu Ala Ala Glu Asn

1 5 10 15

Lys Ser Asn Lys Gln Leu Lys Asp Arg Lys Phe Ser Trp Ala Lys Leu

20 25 30

His Arg Val Asp Ser Leu Asn Leu Glu Ala Gly Lys Val Ser Asn Ser

35 40 45

Ala Ala Gly His Gly Ser Lys Ala Asp Trp Arg Thr Ile Leu Ser Leu

50 55 60

Ala Phe Gln Ser Val Gly Val Ile Tyr Gly Asp Ile Gly Thr Ser Pro

65 70 75 80

Leu Tyr Val Tyr Ala Ser Thr Phe Thr Asp Lys Ile Gly His Lys Asp

85 90 95

Asp Ile Leu Gly Val Leu Ser Leu Ile Ile Tyr Thr Ile Ile Leu Val

100 105 110

Pro Met Thr Lys Tyr Val Phe Ile Val Leu Trp Ala Asn Asp Asn Gly

115 120 125

Asp Gly Gly Ala Phe Ala Leu Tyr Ser Leu Val Cys Arg Tyr Ala Lys

130 135 140

Val Ser Leu Ile Pro Asn Gln Glu Pro Glu Asp Arg Glu Leu Ser His

145 150 155 160

Tyr Asn Leu Glu Leu Pro Ser Asn Gln Phe Arg Arg Ala Gln Lys Ile

165 170 175

Arg His Asn Leu Glu Lys Ser Lys Leu Ala Lys Ile Phe Leu Val Phe

180 185 190

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

195 200 205

Pro Cys Ile Ser Val Leu Ser Ala Val Ser Gly Ile Lys Pro Leu Gly

210 215 220

Gln Glu Ala Ile Val Gly Val Ser Ile Ala Ile Leu Val Ala Leu Phe

225 230 235 240

Cys Val Gln Arg Leu Gly Thr Asp Lys Val Gly Tyr Thr Phe Ala Pro

245 250 255

Ala Ile Cys Ile Trp Phe Leu Phe Ile Ser Gly Ile Gly Leu Tyr Asn

260 265 270

Leu Phe Lys Tyr Asp Val Ser Val Leu Arg Ala Phe Asn Pro Lys Tyr

275 280 285

Ile Ile Asp Tyr Phe Lys Arg Asn Gly Thr Lys Gly Trp Met Ser Pro

290 295 300

Gly Gly Val Phe Leu Cys Ile Thr Gly Ser Glu Ala Met Phe Ala Asp

305 310 315 320

Leu Gly His Phe Ser Val Arg Ser Ile Gln Leu Ser Phe Ser Cys Leu

325 330 335

Val Phe Pro Ala Leu Leu Ser Ala Tyr Ser Gly Gln Ala Ala Tyr Leu

340 345 350

Ser Lys Phe Pro Glu Asp Val Glu Asn Thr Phe Tyr Ala Ser Ile Pro

355 360 365

Asp Pro Leu Tyr Trp Pro Thr Phe Val Val Ala Val Ala Ala Ala Ile

370 375 380

Ile Ala Ser Gln Ala Met Ile Ser Gly Thr Cys Ser Ile Val Ala Gln

385 390 395 400

Ala Gln Gly Leu Gly Cys Phe Pro Arg Val Lys Val Ile His Thr Ser

405 410 415

Ala Lys His Glu Gly Gln Val Tyr Ile Pro Glu Leu Asn Tyr Phe Leu

420 425 430

Met Ile Ala Cys Val Leu Val Thr Leu Ser Phe Lys Thr Thr Glu Lys

435 440 445

Leu Gly His Ala Tyr Gly Ile Ala Val Val Thr Ala Glu Ile Ile Thr

450 455 460

Thr Asn Met Val Thr Leu Val Met Leu Val Ile Trp Lys Ile Ser Ile

465 470 475 480

Trp Trp Ile Thr Leu Phe Tyr Val Val Tyr Phe Ser Ile Glu Ser Thr

485 490 495

Tyr Phe Ser Ala Gln Leu Thr Lys Phe Thr Gln Gly Gly Tyr Leu Pro

500 505 510

Leu Ala Phe Ser Ala Val Leu Val Ile Ile Met Gly Thr Trp His Tyr

515 520 525

Ala Gln Lys Leu Arg Tyr Gln Phe Glu Leu Asn Asn Lys Val Ser Ser

530 535 540

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

545 550 555 560

Gly Ile Gly Leu Leu Tyr Ser Glu Leu Val Gln Gly Ile Pro Pro Ile

565 570 575

Phe Pro His Phe Val Asn Asn Ile Pro Ser Val His Ser Val Ile Val

580 585 590

Leu Val Ser Ile Lys Ser Ile Pro Ile Ser Lys Val Ala Leu Gln Glu

595 600 605

Arg Phe Leu Phe Arg His Val Glu Pro Arg Glu Tyr Asn Val Phe Arg

610 615 620

Cys Val Val Arg Leu Gly Tyr Lys Asp Gln Leu Gly Asp Thr Glu Asp

625 630 635 640

Phe Glu Asn Gln Leu Val Glu Gln Leu Asn Glu Phe Ile Arg His Glu

645 650 655

His Tyr Ile Leu Ala Ala Asp Gln Gln Val Ile Thr Asp Gly Glu Ile

660 665 670

Val Pro Pro Val Ser Gly Gln Leu Val Ser Gly Lys Ser Ser Arg Val

675 680 685

His Met Glu Glu Asp Leu Gln Arg Gln Val Asp Ser Arg Val Ser Ser

690 695 700

Thr Gly Ser Ile Arg Ser Met Asn Thr Pro Ala Ala Gln Ser Asn Arg

705 710 715 720

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

725 730 735

Met Gln Phe Val Lys Lys Ala Lys Gly Gln Gly Val Phe Tyr Leu Leu

740 745 750

Gly Glu Ala Glu Val Val Ala Arg Gln Asp Ala Ser Phe Leu Lys Lys

755 760 765

Ala Phe Val Asn Tyr Gly Tyr Asn Phe Leu Arg Lys Asn Phe Arg Gln

770 775 780

Gly Asp Lys Ala Met Ala Ile Pro Gln Thr Arg Leu Leu Arg Val Gly

785 790 795 800

Met Thr Tyr Glu Leu

805

<210> 2

<211> 2418

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggcgagct cagatagtga acgtcacaca gaagaggctg cagaaaacaa gagcaataaa 60

cagctcaaag atcgcaaatt ctcatgggca aagttacatc gtgttgattc cctcaatttg 120

gaagctggaa aagtttcaaa ctctgcagca ggacacggct ctaaggctga ttggaggacg 180

atactgagtc tagcatttca gtccgtagga gttatctatg gagacatagg gacttcacct 240

ctctacgttt atgcaagtac ttttactgat aaaattggac acaaagatga cattctaggg 300

gtgttgtccc tcatcatata taccataatc ctcgtaccta tgactaagta tgtcttcatt 360

gtcttatggg ccaacgacaa tggcgatggg ggagcatttg cgttgtattc tttagtatgt 420

cgatatgcaa aagtgagtct catcccaaac caagagccag aggacagaga actttcacat 480

tacaatctag agctaccatc taatcaattt agacgagctc agaagatcag acataatttg 540

gaaaagagta aattggcaaa aatatttctt gtcttcgtcg ccatcctcgg aacttctatg 600

gtcattggtg atggagttct cactccttgc atctcagttc tctctgccgt aagtggaata 660

aagcccctag gccaagaggc tattgtgggt gtttccatcg caatactggt agcccttttc 720

tgtgttcaac gattgggtac agacaaggtg ggatatacat ttgctccagc catttgcatt 780

tggtttttgt ttataagtgg cattggcctc tacaatttgt tcaagtatga tgtgagcgtt 840

ttacgtgcct ttaatcccaa atacattatt gactacttca aaagaaacgg tacaaaagga 900

tggatgtctc ctggtggagt tttcctctgc attacaggat ccgaagctat gtttgctgat 960

ttgggtcact tcagtgtgcg ttctattcaa ttaagcttca gttgcctcgt atttccagca 1020

ttgctctcag cttacagtgg acaggctgct tacctctcaa aattccctga agatgttgag 1080

aatactttct atgcctccat cccagatcct ttgtactggc caacatttgt agtggctgtt 1140

gctgcagcca ttattgcaag tcaagcaatg atatctggaa cctgttctat tgtggctcag 1200

gcccaaggtc taggctgttt cccaagggtg aaggtaattc acacatctgc caaacatgag 1260

ggtcaagttt acatcccaga gttgaactac ttcctcatga tcgcatgtgt tctagttact 1320

ctcagcttca aaaccacaga aaaattgggg catgcttatg gtatagctgt ggttactgct 1380

gagattataa ccacaaatat ggtgacacta gtaatgcttg ttatatggaa aatcagcata 1440

tggtggatta ccctattcta cgtggtgtac ttttccatag aatctacata tttttctgcc 1500

cagctgacaa aattcacgca aggtggttat ctacccttag ccttttcagc cgtgctcgtg 1560

atcattatgg gaacttggca ctatgctcaa aaacttcgat accaattcga gctcaataac 1620

aaggtgtcaa gtgagtacgt cagggacttg gctaataatc ctgacatcaa aagagtacct 1680

ggaataggat tactctactc tgaactagtg cagggaattc cacctatatt tcctcatttt 1740

gtcaacaata ttccgtcagt ccattcagtc atagtcctgg tgtcgataaa atccattccc 1800

atcagtaaag tagcactaca ggaacgtttc ttgttccgac acgttgagcc aagagaatac 1860

aatgtgttcc gctgtgtggt taggttggga tacaaagatc aacttgggga tacagaggac 1920

tttgaaaacc aattggtgga acaattgaat gagttcatta gacatgaaca ttacattctt 1980

gcagcagatc aacaagtaat aacagatgga gagattgtac caccagtatc tggacaatta 2040

gtgtccggaa aatcatccag ggtccacatg gaggaagact tacagcgaca agtggattct 2100

agagtttcgt caactggttc aattcgatca atgaacactc cagcagcaca gtccaatcgt 2160

tcatcaagta gaatacaaat ggtgtcccca aatttggaag aagaagagat gcaatttgtg 2220

aaaaaggcaa agggacaagg agtgttctat ctcctgggag aagcagaggt ggtggctaga 2280

caagatgctt cgttcttgaa gaaagcattt gtcaactatg gctataattt cttgcgcaag 2340

aacttcaggc aaggggacaa agctatggct attcctcaaa ctaggctgct aagggtcggc 2400

atgacttatg aactatag 2418

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atggcgagct cagatagtga 20

<210> 4

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

ctatagttca taagtcatgc 20

Claims

1. A tobacco KUP6 protein is characterized in that the amino acid sequence is shown as SEQ ID NO. 1.

2. A gene encoding KUP6 protein of tobacco of claim 1, wherein: the coding region and the nucleotide sequence of the gene are shown as SEQ ID NO. 2.

3. The biomaterial containing the gene of claim 2, wherein the biomaterial is a recombinant expression vector, an expression cassette or a recombinant bacterium.

4. The use of the tobacco KUP6 protein or its coding gene of claim 1 or the biomaterial of claim 3 for promoting the absorption and transport of potassium ions by microorganisms, which are yeasts.