CN109354612B - Tobacco AKT2/3 gene and application thereof - Google Patents
Tobacco AKT2/3 gene and application thereof Download PDFInfo
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- CN109354612B CN109354612B CN201811339759.7A CN201811339759A CN109354612B CN 109354612 B CN109354612 B CN 109354612B CN 201811339759 A CN201811339759 A CN 201811339759A CN 109354612 B CN109354612 B CN 109354612B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
Abstract
The invention relates to a tobacco AKT2/3 gene and application thereof. The tobacco AKT2/3 gene and the sequence of the protein coded by the gene are respectively shown in SEQ ID NO. 1 and SEQ ID NO. 2. The invention clones the AKT2/3 gene from tobacco for the first time, verifies the biological function of the gene through a yeast function complementation experiment, and the tobacco AKT2/3 gene has the function of promoting potassium ion absorption and transfer.
Description
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:
extracting total RNA of tobacco cells before AKT2/3 gene PCR amplification, and reversely transcribing the extracted total RNA into cDNA. In the present invention, the extraction of the total RNA of the tobacco cells may be performed by a technical scheme commonly used in the art for extracting the total RNA of the cells, and in the embodiment of the present invention, a Trizol method may be specifically used. In the invention, the raw material for extracting the total RNA of the tobacco cells is fresh leaves of tobacco, and the tobacco is a tobacco variety conventional in the field, such as K326.
And secondly, after the total RNA of the tobacco cells is extracted, carrying out reverse transcription on the total RNA of the tobacco cells to synthesize cDNA. In the invention, the cDNA is synthesized by adopting a conventional cDNA synthesis method in the field without other special requirements; in the specific embodiment of the present invention, cDNA synthesis was performed using a cDNA synthesis kit available from TaKaRa.
③ after obtaining the cDNA, the AKT2/3 gene PCR amplification is carried out to obtain the target fragment. In the present invention, the AKT2/3 gene PCR amplification system is preferably 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, 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.
After the target fragment is obtained by the PCR amplification of the AKT2/3 gene, sequencing the target fragment to obtain the AKT2/3 gene. The present invention preferably purifies the target fragment after the PCR amplification, and the purification method of the present invention is not particularly limited, and may be performed using a DNA purification kit well known to those skilled in the art.
Fifthly, after the purification is finished, preferably, the purified target fragment is introduced into escherichia coli DH5 alpha competent cells for colony PCR, and the result is verified to be positiveSex cloning was followed by sequencing. After obtaining positive clones, the invention preferably adopts a colony PCR method to verify the positive clones. In the present 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 colony PCR system is 10 muL, including 5 muL of Premix ExTaq, 0.5 muL of 10 muM forward primer, 0.5 muL of 10 muM reverse primer, ddH2O4. mu.L. In the present invention, the method for introducing the purified target fragment into E.coli DH5 alpha competent cells is a method for transforming E.coli competent cells, which is conventional in the art, and specifically comprises the following steps: connecting the target fragment with a pMD19-T vector for 10-14 h at 16 ℃ to obtain a connection product; transforming the ligation product into escherichia coli DH5 alpha competent cells to obtain transformed escherichia coli DH5 alpha; the transformed E.coli DH5 alpha was inoculated on LB plate coated with ampicillin to perform screening culture, and positive clones were obtained.
Sixthly, after the 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; alternatively, the first and second electrodes may be,
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 leaves (the tobacco variety is K326), extracting total RNA of tobacco cells by adopting a Trizol method, then synthesizing cDNA by adopting a cDNA synthesis kit of TaKaRa company, further adopting Primer5.0 software design 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'-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 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 positive clones, verifying the positive clones by adopting a colony PCR method, wherein forward primers of the colony PCR are as follows: 5'-ATGCAAATGAGCCATTCATC-3', respectively; the reverse primer is 5'-CTACAAGCAATTTGGATCT-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 3 independent positive clones are randomly selected from the verified positive clones and sent to a biotechnology company for sequencing, and the sequence of the tobacco AKT2/3 gene obtained by sequencing is shown as SEQ ID NO. 1.
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 M and Sanders D1996 mechanics of potassium adsorption by highher plant roots. 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 respectively subjected to double enzyme digestion (enzyme digestion sites are Xba I and Xho I), 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 alpha, and PCR amplification and enzyme digestion are carried out on a single colony of the transformed Escherichia coli 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
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Claims (4)
1. The amino acid sequence of the tobacco AKT2/3 gene coded protein is shown in 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 yeast potassium ion absorption and transport.
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| CN201811339759.7A CN109354612B (en) | 2018-11-12 | 2018-11-12 | Tobacco AKT2/3 gene and application thereof |
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