CN109336958A

CN109336958A - A kind of potassium transport protein KUP10 and its encoding gene and application from tobacco

Info

Publication number: CN109336958A
Application number: CN201811341857.4A
Authority: CN
Inventors: 张洁; 任学良; 李丽芹; 鲁黎明; 王仁刚; 王自力; 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: 2019-02-15
Anticipated expiration: 2038-11-12
Also published as: CN109336958B

Abstract

The present invention relates to genetic engineering field, a kind of potassium transport protein KUP10 from tobacco and its encoding gene and application are specifically disclosed.The present invention provides the KUP10 gene for being isolated from tobacco for the first time, the KUP10 full length gene 2532bp, through functional verification, after KUP10 gene provided by the invention is transferred to potassium native defect type yeast mutant R5421, the recombination yeast for expressing the KUP10 gene is provided with the function of Potassium Absorption and transhipment again.Therefore, KUP10 gene provided by the invention has the function of promoting Potassium Absorption and transhipment.

Description

A kind of potassium transport protein KUP10 and its encoding gene and application from tobacco

Technical field

The present invention relates to genetic engineering fields, specifically, be related to a kind of potassium transport protein KUP10 from tobacco and its Encoding gene and application.

Background technique

Potassium transporter is can be when extraneous potassium concentration be extremely low by kalium ion transport to intracellular carrier protein.Potassium turns Transport body be usually some transmembrane proteins, by conformation change complete potassium ion transmembrane transport, be it is a kind of need ATP provide energy Active transport processes, usually K⁺/H⁺Or K⁺/Na⁺Collaboration transport.

The prior art for potassium transporter gene research in model plant arabidopsis than wide, for example, research table Bright Arabidopsis Mutants kup6 is reduced compared with survival rate of the WT lines under drought stress, and 35S::KUP6 overexpression turns base Because the drought-resistance ability of plant has but obtained apparent enhancing (Shabala et al., 2007)；And the mutant children of KUP7 gene Seedling shows the low potassium sensitive phenotype of yellowing leaf under low potassium stress, and the potassium content of root is remarkably decreased, KUP7 base Because the missing of function reduces Arabidopsis thaliana Seedlings under the conditions of low potassium to the absorbability of potassium, potassium concentration in xylem sap It reduces (Min et al., 2016).

Tobacco is a kind of crop that consumption potassium amount is very big, and potassium content of tobacco leaf is the important indicator for measuring quality of tobacco, currently, It is less for the research of Potassium in Tobacco transporter gene, the Unknown Function of tobacco KUP.

Summary of the invention

In order to solve the problems in the existing technology, the object of the present invention is to provide a kind of, and the potassium from tobacco transports egg White KUP10 and its encoding gene and application.

In order to achieve the object of the present invention, technical scheme is as follows:

Present invention firstly provides a kind of potassium transport protein, it is obtained from tobacco, is named as KUP10 albumen, is following (a) or (b)

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

(b) sequence shown in SEQ ID NO.1 by the substitution of one or several amino acid residues and/or missing and/or adds Add and to plant Potassium Absorption and the relevant protein as derived from (a) of transhipment regulation.

KUP10 albumen in above-mentioned (b) can be artificial synthesized, can also first synthesize its encoding gene, then carries out biological expression and obtain It arrives.The encoding gene of KUP10 albumen in above-mentioned (b) can by will in DNA sequence dna shown in SEQ ID NO.2 lack one or The codon of several amino acid residues, and/or carry out the missense mutation of one or several base-pairs and obtain.

The gene (KUP10 gene) for encoding the KUP10 albumen also belongs to protection scope of the present invention.

The gene concretely following DNA molecular 1) or 2) or 3):

1) code area DNA molecular as shown in SEQ ID NO.2；

2) hybridize under strict conditions with the DNA sequence dna 1) limited and encode and plant Potassium Absorption and transhipment regulation phase Close the DNA molecular of albumen；

1) or 2) 3) with the DNA sequence dna limited there is 90% or more homology and coding and plant Potassium Absorption and transhipment Regulate and control the DNA molecular of GAP-associated protein GAP.

Above-mentioned stringent condition can be for 0.1 × SSPE (or 0.1 × SSC), the solution of 0.1%SDS be miscellaneous in DNA or RNA It hands over and hybridizes at 65 DEG C in experiment and wash film.

It has been investigated that the KUP10 gene plays obvious effect in terms of promoting Potassium Absorption and transhipment.

Further, KUP10 gene of the present invention is prepared by the following steps to obtain:

(1) PCR amplification primer is designed, the PCR amplification primer includes forward primer and reverse primer:

Forward primer: 5 '-ATGGGGAAAGGAGAGATAGA-3 ',

Reverse primer: 5 '-TCAAACCATGTATGTCATTC-3 '；

(2) tobacco cell total serum IgE is extracted, tobacco cell cDNA is synthesized, carries out KUP10 base by template of tobacco cell cDNA The PCR amplification of cause obtains target fragment, sequencing.

In one embodiment of the invention, the system of the PCR amplification is 20 μ L systems, including Premix ExTaq 10 μ L, 10 μM of forward primer 0.5 μ L, 10 μM of reverse primer 0.5 μ L, tobacco cell cDNA 1 μ L, ddH₂O 8μL。

Preferably, the response procedures of the PCR amplification are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s；55 DEG C of annealing 30s；72 DEG C of extension 2min；35 circulations.

Preferably, the target fragment further includes that the target fragment is imported bacillus coli DH 5 alpha before sequencing Bacterium colony PCR verifying is carried out in competent cell, is sequenced after being verified as positive colony.

Preferably, the bacterium colony PCR verifies the forward primer nucleotide sequence used are as follows: 5 '- ATGGGGAAAGGAGAGATAGA-3 ', bacterium colony PCR verify the reverse primer nucleotide sequence used are as follows: 5 '- TCAAACCATGTATGTCATTC-3’。

Preferably, the system of the bacterium colony PCR verifying is 10 μ L, including 5 μ L of Premix ExTaq, 10 μM of forward direction are drawn Object 0.5 μ L, 10 μM of reverse primer 0.5 μ L, ddH₂O 4μL。

Further, recombinant expression carrier, expression cassette, transgenic cell line or recombinant bacterium containing the KUP10 gene Equal biomaterials all belong to the scope of protection of the present invention.

The recombinant expression carrier can be constructed with existing plant expression vector.The recombinant expression carrier for example may be used To be double base agrobacterium vector and the carrier etc. that can be used for plant micropellet bombardment.Use the gene constructed recombinant expression carrier of KUP10 When, any enhanced, composing type, organizing specific type or inducible promoter can be added before its transcription initiation nucleotide, They can be used alone or are used in combination with other plant promoters；In addition, using the gene constructed recombinant expression carrier of KUP10 When, also enhancer, including translational enhancer or transcriptional enhancer, these enhancer regions can be used to can be ATG initiation codon Son or neighboring region initiation codon etc., but must be identical as the reading frame of coded sequence, to guarantee correctly turning over for entire sequence It translates.The source of the translation control signal and initiation codon be it is extensive, can be natural, be also possible to synthesis.It turns over Translating initiation region can come from transcription initiation region or structural gene.

The recombinant expression carrier for carrying the KUP10 gene can be by Ti-plasmids, Ri plasmid, plant viral vector, straight The conventional biology methods such as DNA conversion, microinjection, conductance, mediated by agriculture bacillus are connect to be transformed into plant cell or tissue.

Further, the present invention also protects the KUP10 albumen, the KUP10 gene and the life containing the gene Object material is promoting the application in plant or microorganism potassium Ions Absorption and transhipment.

The plant includes tobacco, arabidopsis, and the microorganism includes saccharomycete.

For example, in a specific embodiment of the invention, the KUP10 gene is transferred to potassium native defect type yeast mutation After strain R5421, the recombination yeast for expressing the KUP10 gene is provided with the function of Potassium Absorption and transhipment again；By tobacco After KUP10 gene described in plant carries out overexpression, it is remarkably improved the content of potassium ion in the tobacco leaf of tobacco plant.

The application is chosen as, and aforementioned tobaccos KUP10 gene is transferred in tobacco plant, makes KUP10 gene excess table Up to the content of potassium ion in the tobacco leaf to improve tobacco plant.

Further, the present invention also protects the KUP10 albumen, the KUP10 gene and the life containing the gene Application of the object material in prepare transgenosis plant.

Further, the present invention also protects the KUP10 albumen, the KUP10 gene and the life containing the gene Application of the object material in plant breeding.

The purpose of the breeding is to promote plant Potassium Absorption and transhipment, preferably potassium in the tobacco leaf of raising tobacco plant The content of ion.

The plant is monocotyledon or dicotyledon.Such as tobacco or arabidopsis KUP10 mutant strain.

The beneficial effects of the present invention are:

The present invention provides the KUP10 gene for being isolated from tobacco, the KUP10 full length gene 2532bp, through function for the first time Verifying, after KUP10 gene provided by the invention is transferred to potassium native defect type yeast mutant R5421, expresses the KUP10 gene Recombination yeast be provided with the function of Potassium Absorption and transhipment again.Therefore, KUP10 gene provided by the invention, which has, promotes The function of Potassium Absorption and transhipment.

Detailed description of the invention

Fig. 1 is yeast function complementation experiment result in the embodiment of the present invention 2.Wherein, A is the training that potassium concentration is 20uM The growing state on base is supported, B is that potassium concentration is growing state on the culture medium of 2mM；In figure, 1 is to be transferred to KUP10 gene Recombination yeast, 2 be negative control (being transferred to empty carrier), and 3 are transferred to the yeast of arabidopsis KUP gene for positive control；From a left side to The right side is followed successively by the growth result of stoste, 10 times of dilutions, 100 times of dilutions on culture medium.

Specific embodiment

The preferred embodiment of the present invention is described in detail below in conjunction with embodiment.It will be appreciated that following real Providing merely to play the purpose of explanation for example is applied, is not used to limit the scope of the present invention.The skill of this field Art personnel without departing from the spirit and purpose of the present invention, can carry out various modifications and replace to the present invention.

Experimental method used in following embodiments is conventional method unless otherwise specified.Institute in following embodiments Material, reagent etc., are commercially available unless otherwise specified.

Potassium native defect type yeast mutant R5421 of the invention is recorded in Maathuis F J M and Sanders D 1996Mechanisms of potassium absorption by higher plant roots.Physiol.Plant.96,158–168.P416 yeast sequestered shuttle expression carrier, TEF constitutive promoter, CYC1 terminator, CEN6ARSH4 replication orgin, selection markers are URA3 in yeast, and selection markers are Amp in Escherichia coli.Note It is loaded in Functional Expression of a ω -3Fatty Acid Desaturase Gene from Glycine max in Saccharomyces cerevisiae。

The acquisition of 1 KUP10 gene of embodiment

The fresh blade of 0.5g tobacco is taken, the total serum IgE of tobacco cell is extracted using Trizol method, then uses TaKaRa company CDNA synthetic agent box synthesize cDNA, further obtain primer using Primer5.0 software design and by artificial optimization, institute Stating primer includes forward primer and reverse primer, the forward primer nucleotide sequence are as follows: 5 '- ATGGGGAAAGGAGAGATAGA-3'；The nucleotides sequence of the reverse primer is classified as 5 '-TCAAACCATGTATGTCATTC-3 ', Using the cDNA of synthesis as template, PCR amplification is carried out.

PCR amplification system is 20 μ L systems, comprising: Premix ExTaq 10 μ L, 10 μM of forward primer 0.5 μ L, 10 μM Reverse primer 0.5 μ L, tobacco cell cDNA 1 μ L, ddH₂O 8μL。

Pcr amplification reaction program are as follows: 95 DEG C of initial denaturation 5min；95 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend 2min, 35 circulations.

After the completion of PCR amplification, the purifying of target fragment is carried out using DNA purification kit, by target fragment after purification 12h is connect with 16 DEG C of pMD19-T carrier and obtains connection product, by the obtained connection product conversion bacillus coli DH 5 alpha impression State cell converted after bacillus coli DH 5 alpha, the bacillus coli DH 5 alpha after the conversion is inoculated in and is coated with ammonia benzyl mould Screening and culturing is carried out on the LB plate of element obtains positive colony.After obtaining positive colony, verified using the method for bacterium colony PCR Positive colony, the forward primer of the bacterium colony PCR are as follows: 5 '-ATGGGGAAAGGAGAGATAGA-3 ', reverse primer are as follows: 5 '- TCAAACCATGTATGTCATTC-3'；The system of the bacterium colony PCR is 10 μ L, including 5 μ L of Premix ExTaq, and 10 μM just To primer 0.5 μ L, 10 μM of reverse primer 0.5 μ L, ddH₂O 4μL.Then 3 are randomly selected from the positive colony having verified that Independent positive colony is sent to biotech company and is sequenced, and the present invention obtains the sequence of the KUP10 gene after sequencing Column, as shown in SEQ ID NO.2.

Effect of the embodiment 2KUP10 gene in terms of promoting Potassium Absorption and transhipment

The T- carrier for being connected with KUP10 gene described in embodiment 1 and expression vector P416 are subjected to double digestion respectively (restriction enzyme site are as follows: Sma I and Xho I) recycles target gene and expression vector P416, is then connected with ligase, after connection Recombinant yeast expression vector be transferred to the competent cell of bacillus coli DH 5 alpha, the Escherichia coli single colonie after conversion is carried out PCR amplification, digestion verify whether to construct successfully, will construct successful recombinant yeast expression vector and are transferred in R5421.

Specific step is as follows: being lined on solid medium YPDA with the collarium R5421 saccharomycete deposited of going bail for is connect, 28 DEG C of trainings Support 12h；Picking R5421 yeast single bacterium is fallen in Ep pipe, and 1mL YPDA culture solution is added to be vortexed；Above-mentioned bacterium solution is all transferred to and is equipped with In the triangular flask of YPDA culture solution, in 30 DEG C, 250rpm shakes bacterium to OD600=1.2；It transfers, is shaken to OD600=1.0 by 1:10 ~1.2；In 28 DEG C, 1000rpm is centrifuged 5min collection bacterium, is resuspended with the sterilizing ultrapure water of 1/2 volume；In 28 DEG C, 1000rpm is centrifuged 5min collection bacterium, blots supernatant；Sequentially add following ingredients (every original bacterium solution of 5mL):

Vortex 1min, mixes transformation system completely；It is put in 30 DEG C of water-bath and incubates 30min；Place into 42 DEG C of water Thermal shock 28min in bath, cooled on ice 10min；7000rpm is centrifuged 15s, abandons supernatant；Precipitating is gently resuspended with the sterile water of 1mL； 200 μ L transformation mixtures are taken to be laid on auxotroph plate；30 DEG C are cultivated 3 days.Extract yeast plasmid and qualification result.

In the flat lining out of auxotrophy, 30 DEG C are cultivated 3 days the yeast single colonie that picking was identified；It is lacked with toothpick in nutrition A small amount of thallus, which is dipped, on sunken plate cultivates 12h in 2mL auxotrophy liquid；8000rpm is centrifuged 1min and collects thallus；Supernatant is abandoned, With 1mL distilled water suspension thalline, 8000rpm is centrifuged 1min；

Supernatant is abandoned, with 1mL distilled water resuspension, adjusts OD₆₀₀It is 0.8；10 times, 100 times are diluted by undiluted bacterium solution and respectively Bacterium solution afterwards, taking 5uL respectively in potassium concentration is 20uM, on the culture medium of 2mM, 30 DEG C of cultures, 3 days observation results.

Experimental result is as shown in Figure 1, be 20uM, 2mM culture medium (AP culture medium (1L): 546 μ of phosphoric acid in potassium concentration L, L-arginine 1.742g, 1000 × vitamin solution 1mL, 1000 × trace element solution 1mL, uracil 0.77g, 100 × Ura 10mL, glucose 20g, agar powder 15g) on, negative control group saccharomycete (being transferred to P416 empty carrier) is hardly grown, and is turned The recombinant yeast of the recombination yeast and positive controls (being transferred to arabidopsis KUP gene) that enter tobacco KUP10 gene can give birth to It is long.With the increase of extension rate, the recombinant yeast of the recombination yeast and positive controls that are transferred to tobacco KUP10 gene is still It can grow.Result above proves that tobacco KUP10 gene of the invention has potassium absorption and transport function.

Although above the present invention is described in detail with a general description of the specific embodiments, On the basis of the present invention, it can be made some modifications or improvements, this will be apparent to those skilled in the art.Cause This, these modifications or improvements, fall within the scope of the claimed invention without departing from theon the basis of the spirit of the present invention.

Sequence table

<110>Guizhou Province Tabacco Science and Technology Institute

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

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Ser Leu Gly Ser Ile Gly Ile Tyr Asn Leu Leu Lys His Asp Val Thr

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Ser Glu Val Lys Gln Lys Ile Ser Met Asp Phe Met His Glu Leu Gly

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Leu Pro Ala Ile His Ser Val Ile Val Phe Val Cys Ile Lys Tyr Val

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Pro Val Pro Ile Val Pro Gln Asp Glu Arg Phe Leu Phe Arg Arg Val

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Gly Pro Lys Asp Tyr His Met Phe Arg Cys Val Ala Arg Tyr Gly Tyr

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Claims

1. a kind of KUP10 protein from tobacco, which is characterized in that be following (a) or (b):

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

(b) sequence shown in SEQ ID NO.1 by one or several amino acid residues substitution and/or deletion and/or addition and The protein as derived from (a) relevant to plant Potassium Absorption and transhipment regulation.

2. encoding the gene of protein described in claim 1, which is characterized in that the gene is following DNA 1) or 2) or 3) Molecule:

1) code area DNA molecular as shown in SEQ ID NO.2；

2) under strict conditions to the DNA sequence dna hybridization and coding that 1) limit and plant Potassium Absorption and transport the related egg of regulation White DNA molecular；

1) or 2) 3) with the DNA sequence dna limited there is 90% or more homology and coding and plant Potassium Absorption and transhipment regulation The DNA molecular of GAP-associated protein GAP.

3. the biomaterial containing gene described in claim 2, the biomaterial is recombinant expression carrier, expression cassette, turns base Because of cell line or recombinant bacterium.

4. protein described in claim 1 or gene as claimed in claim 2 or biomaterial as claimed in claim 3 exist Promote the application in plant or microorganism potassium Ions Absorption and transhipment.

5. application according to claim 4, which is characterized in that the plant includes tobacco, arabidopsis.

6. application according to claim 4, which is characterized in that the microorganism includes saccharomycete.

7. protein described in claim 1 or gene as claimed in claim 2 or biomaterial system as claimed in claim 3 Application in standby genetically modified plants.

8. protein described in claim 1 or gene as claimed in claim 2 or biomaterial as claimed in claim 3 exist Application in plant breeding.

9. application according to claim 8, which is characterized in that the purpose of the breeding be promote plant Potassium Absorption and Transhipment.

10. application according to claim 8, which is characterized in that the plant is tobacco.