CN109485709A

CN109485709A - A kind of tobacco KUP4 albumen and its encoding gene and application

Info

Publication number: CN109485709A
Application number: CN201811339649.0A
Authority: CN
Inventors: 王自力; 任学良; 李丽芹; 鲁黎明; 王仁刚; 张洁; 耿召良
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: 2019-03-19
Anticipated expiration: 2038-11-12
Also published as: CN109485709B

Abstract

The present invention relates to genetic engineering field, a kind of tobacco KUP4 albumen and its encoding gene and application are specifically disclosed.The present invention provides the KUP4 gene for being isolated from tobacco for the first time, the KUP4 gene nucleotide series are as shown in SEQ ID NO.2, the amino acid sequence of its albumen KUP4 encoded is as shown in SEQ ID NO.1, through functional verification, after KUP4 gene provided by the invention is transferred to potassium native defect type yeast mutant R5421, the recombination yeast for expressing the KUP4 gene has Potassium Absorption, transport function.Illustrate that KUP4 gene provided by the invention has the function of promoting Potassium Absorption and transhipment.

Description

A kind of tobacco KUP4 albumen and its encoding gene and application

Technical field

The present invention relates to genetic engineering fields, specifically, be related to a kind of potassium transport protein KUP4 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 tobacco KUP4 albumen and its volumes Code 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 KUP4 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 there is the protein as derived from (a) with (a) same function.

The present invention provides the gene for encoding the tobacco KUP4 albumen, the gene is following DNA 1) or 2) or 3) Molecule:

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.

It has been investigated that the KUP4 gene plays obvious effect in terms of promoting Potassium Absorption and transhipment.

Further, KUP4 gene of the present invention is prepared by the following steps to obtain:

PCR amplification primer is designed, the PCR amplification primer includes forward primer and reverse primer: the nucleosides of forward primer Acid sequence are as follows: the nucleotides sequence of 5 '-ATGGATCTTGAAACAGGGTT-3 ', the reverse primer are classified as 5 '- TCATACATGATAGACCATTC-3'；(2) tobacco cell total serum IgE is extracted, tobacco cell cDNA is synthesized, with tobacco cell cDNA The PCR amplification that KUP4 gene is carried out for template, obtains target fragment, is sequenced.

Preferably, the system of the PCR amplification is 20 μ L systems, including Premix ExTaq10 μ L, 10 μM of forward direction 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 '- ATGGATCTTGAAACAGGGTT-3 ', bacterium colony PCR verify the reverse primer nucleotide sequence used are as follows: 5 '- TCATACATGATAGACCATTC-3’。

Preferably, the system of the bacterium colony PCR verifying is 10 μ L, including Premix ExTaq5 μ L, 10 μM of forward primer 0.5 μ L, 10 μM of reverse primer 0.5 μ L, ddH₂O 4μL。

The present invention provides the biomaterial of the gene containing above-mentioned encoding nicotiana KUP4 albumen, the biomaterial is attached most importance to Group expression vector, expression cassette, transgenic cell line or recombinant bacterium.

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 KUP4 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 KUP4 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.

Carry the KUP4 gene recombinant expression carrier can by Ti-plasmids, Ri plasmid, plant viral vector, directly The conventional biology methods such as DNA conversion, microinjection, conductance, mediated by agriculture bacillus are transformed into plant cell or tissue.

Biomaterial the present invention provides above-mentioned tobacco KUP4 albumen or its encoding gene or containing the gene is promoting Application in plant or microorganism potassium Ions Absorption and transhipment.

The plant includes tobacco, arabidopsis.

The microorganism includes yeast.

For example, in a specific embodiment of the invention, the KUP4 gene is transferred to potassium native defect type yeast mutation After strain R5421, the recombination yeast for expressing the KUP4 gene is provided with the function of Potassium Absorption and transhipment again；Tobacco is planted After KUP4 gene described in strain 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 KUP4 gene is transferred in tobacco plant, makes KUP4 gene overexpression The content of potassium ion in tobacco leaf to improve tobacco plant.

It is prepared by the biomaterial the present invention provides above-mentioned tobacco KUP4 albumen or its encoding gene or containing the gene Application in genetically modified plants.

Biomaterial the present invention provides above-mentioned tobacco KUP4 albumen or its encoding gene or containing the gene is in plant Application in breeding.

The purpose of the breeding is to promote or adjust plant Potassium Absorption and transhipment.

The plant is tobacco.

Provide the KUP4 gene for being isolated from tobacco, the KUP4 base for the first time the beneficial effects of the present invention are: the present invention Because nucleotide sequence is as shown in SEQ IDNO.2, the amino acid sequence of albumen is encoded as shown in SEQ ID NO.1.It is tested through function Card, KUP4 gene provided by the invention are transferred to potassium native defect type yeast mutant R5421 (Manthuis FJM, Sander SD.Mechanisms of potassium absorption by higher plant roots.Physiol Plant, 1996,96:158-168) after, the recombination yeast for expressing the KUP4 gene is provided with the function of Potassium Absorption and transhipment again Energy.Illustrate that KUP4 gene provided by the invention has the function of promoting Potassium Absorption and transhipment.

Detailed description of the invention

Figure 1A and Figure 1B be respectively in embodiment 2 KUP gene be transferred to the weight after potassium native defect type yeast mutant R5421 Growing state of the group yeast on the culture medium that potassium concentration is 20uM and 2mM；In figure, 1 is negative control P416, and 2 be potassium Transporter (recombination yeast for being transferred to KUP4 gene), 3 are transferred to the saccharomycete of arabidopsis KUP gene for positive control；From left to right It is followed successively by stoste, 10 times of dilutions, 100 times of dilutions, 1000 times of dilutions；Stoste, 10 times are from left to right followed successively by Figure 1B The growth result of dilution, 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.

The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.

The acquisition of 1 KUP4 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 '- ATGGATCTTGAAACAGGGTT-3 ', length 20bp；The nucleotides sequence of the reverse primer is classified as 5 '- TCATACATGATAGACCATTC-3 ', length 20bp.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, ddH₂O8μL。

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 '-ATGGATCTTGAAACAGGGTT-3 ', reverse primer are as follows: 5 '- TCATACATGATAGACCATTC-3'；The system of the bacterium colony PCR is 10 μ L, including 5 μ L of Premix ExTaq, and 10 μM just To 0.5 μ L of primer, 10 μM of reverse primer, 0.5 4 μ L of μ L, ddH2O.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 KUP4 gene after sequencing Column, as shown in SEQ ID NO.2, amino acid sequence is as shown in SEQ IDNO.1.

Effect of the 2 KUP4 gene of embodiment in terms of promoting Potassium Absorption and transhipment

The T- carrier and expression vector P416 (p416 yeast sequestered of KUP4 gene described in embodiment 1 will be connected with Shuttle expression carrier, TEF constitutive promoter, CYC1 terminator, CEN6ARSH4 replication orgin, selection markers are in yeast URA3, selection markers are Amp in Escherichia coli.Select from Functional Expression of a ω -3Fatty Acid Desaturase Gene from Glycine max in Saccharomyces cerevisiae) double digestion is carried out respectively (restriction enzyme site are as follows: SmaI and XhoI) 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 FIG. 1A and 1B, is 20uM, 2mM culture medium (AP culture medium (1L): phosphorus in potassium concentration Acid 546 μ 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 raw It is long, it is transferred to the recombination yeast of tobacco KUP4 gene and the recombinant yeast of positive controls (being transferred to arabidopsis KUP gene) With growth.With the increase of extension rate, it is transferred to the recombination yeast of tobacco KUP4 gene and the recombinant yeast of positive controls Still it can grow.

The above results prove that KUP4 gene of the present 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

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Claims

1. a kind of tobacco KUP4 albumen, 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 With the protein as derived from (a) with (a) same function.

2. encoding the gene of tobacco KUP4 albumen described in claim 1, it is characterised in that: the gene be it is following 1) or 2) or 3) DNA molecular:

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. tobacco KUP4 albumen or its encoding gene described in claim 1 or biomaterial as claimed in claim 3 are promoting to plant Application in object 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 yeast.

7. tobacco KUP4 albumen or its encoding gene described in claim 1 or biomaterial as claimed in claim 3 turn in preparation Application in gene plant.

8. tobacco KUP4 albumen or its encoding gene described in claim 1 or biomaterial as claimed in claim 3 are educated in plant Application in kind.

9. application as claimed in claim 8, which is characterized in that the purpose of the breeding is to promote or adjust plant potassium ion to inhale It receives and transports.

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