CN114457090A - Mangifera indica root potassium ion channel gene MiSKOR as well as construction method and application of expression vector - Google Patents

Abstract

The invention discloses a mango root potassium channel geneMiSKORMangifera indica K, expression vector construction method and application⁺Channel geneMiSKORThe base sequence is shown as SEQ ID NO: 1, and the coded product is Shaker K⁺The channel protein has an amino acid sequence shown as SEQ ID NO: 2, and also discloses a recombinant expression vector pTracer-CMV3-MiSKORAnd pHB-MiSKORThe method of (1). The gene is specially used for root of grafted seedling of mangoThe heteroexpression has the characteristics of sensitively responding to the external potassium supply level and mediating K⁺Electrophysiological functions of outward flow and transport; after the gene is overexpressed in arabidopsis thaliana, the potassium enrichment capacity and NaCl stress tolerance of transgenic arabidopsis thaliana are improved, and the gene has important application value in the aspects of improving the high-efficiency transportation and stress resistance of crop potassium.

Description

Mangifera indica root potassium ion channel gene MiSKOR as well as construction method and application of expression vector

Technical Field

The invention relates to a mango root potassium ion channel gene, a construction method of an expression vector and application.

Background

Potassium ion (K)⁺) Is the most abundant mineral nutrient element in plant cells, maintains the charge balance of the cells, regulates the osmotic potential of the cells, participates in various life processes such as stomata opening and closing, photosynthesis, transpiration and the like, and is closely related to the growth, development and fruit quality of the plants. In horticulture, the potassium deficiency of soil seriously inhibits the growth and development of fruit trees, and affects the quality, yield and storage performance after picking, and many researches are mainly embodied in the physiological and biochemical level, and the molecular mechanism research of the high efficiency and utilization of potassium nutrition of fruit trees is scarce.

Plants need to take up the required K from the soil through the roots⁺And then distributed to different organ sites to meet normal growth and development. Plant K⁺There are 2 mechanisms of nutrient absorption: mechanism I, high affinity potassium absorption system, K + concentration less than 200 μmol. L in external environment^-1The active potassium absorption process which plays a main role; mechanism II, low affinity potassium absorption System, in the external Environment K⁺The concentration is more than 1 mmol.L^-1The dominant passive potassium uptake process (Epstein et al, 1963; Very et al, 2003). In vivo K of plants⁺Long distance transport and distribution is caused by various types of K localized to the cytoplasmic membrane⁺The channel mediated process can be divided into Shaker family, TPK family and other Ks according to the difference of channel protein sequence, structure and function⁺Channel 3 is of a major class (Lebaudy et al, 2007). Wherein the Shaker class K⁺The study of the channel is the mostTo a substrate K⁺Has an affinity constant of tens of millimoles and belongs to the typical low-affinity and high-flux K⁺A channel. According to voltage dependence and K⁺The difference in direction of motion in the case of transmembrane, Shaker type K⁺The channels may be further divided into inward rectifying, outward rectifying, and weak-direction rectifying (i.e., bidirectional rectifying) channels. Wherein, SKOR codes are outward rectifier type Shaker type K⁺The channel plays an important role in the aspect of high nutrition and high efficiency of plant potassium.

At present, only grape SKOR research reports (Shenjing Yuan and the like, 2020) exist in fruit science, and no report related to SKOR channel exists in tropical fruit crops. Therefore, the novel SKOR homologous gene is separated and identified from the mango trees, theoretical basis and gene materials can be provided for researching the molecular mechanism of high potassium nutrition and high efficiency of tropical fruit tree crops, and the method has important significance.

Disclosure of Invention

The invention aims to provide a mango root potassium channel gene MiSKOR and a construction method of an expression vector for efficiently expressing the MiSKOR gene, a preparation method of transgenic arabidopsis thaliana with MiSKOR heterologous overexpression and application of the gene in the aspects of improving the high efficiency and stress resistance of plant potassium nutrition.

The technical solution of the invention is as follows:

a mango root potassium channel gene MiSKOR has a base sequence shown in SEQ ID NO: 1, and the following components:

a construction method of a recombinant expression vector pTracer-CMV3-MiSKOR of a mango root potassium channel gene MiSKOR is characterized in that: designing a specific primer for constructing a recombinant plasmid, wherein a 5 'end primer is introduced into a Kpn I enzyme cutting site, the base sequence is GAGGGTACCA TGGGCGATAC TGCAAGAGAG GA, a 3' end primer is introduced into a Not I enzyme cutting site, the base sequence is GAAGCGGCCG CTTACAAGCT CTTCAATGGT ATG, amplifying and cloning a CDS fragment of a mango MiSKOR gene by a PCR method, and recovering a PCR product; PCR recovery products of MiSKOR genes and pTracer-CMV3 plasmids are respectively cut by Kpn I and Not I, then T4 DNA ligase is used for connection, the connection products are transformed into escherichia coli, positive clones are screened, plasmids are extracted by a plasmid minikit, and the recombinant expression vector pTracer-CMV3-MiSKOR is obtained after sequencing verification.

A method for creating a transgenic arabidopsis thaliana strain with a mango root potassium ion channel gene MiSKOR heterogeneously and excessively expressed is characterized in that: comprises the following steps:

(1) expression vector pHB-MiSKOR construction

Designing a specific primer for constructing a recombinant plasmid, wherein a 5 'end primer introduces a BamH I restriction site with a base sequence of GAGGGATCCA TGGGCGATAC TGCAAGAGAG GA, a 3' end primer introduces an XbaI restriction site with a base sequence of GCGTCTAGAT TACAAGCTCT TCAATGGTAT GC, amplifying a Cerbera Manghas MiSKOR gene CDS fragment by a PCR method, and recovering a PCR product; respectively carrying out double digestion on a PCR (polymerase chain reaction) recovery product of the MiSKOR gene and a pHB plasmid by using BamH I and XbaI, then connecting the products by using T4 DNA ligase, transforming escherichia coli by using the connection product, screening positive clones, extracting the plasmid by using a plasmid miniprep kit, and carrying out sequencing verification to obtain a correct recombinant expression vector pHB-MiSKOR;

(2) generation of transgenic Arabidopsis with overexpression

The constructed recombinant expression vector pHB-MiSKOR was infected into the floral foam of wild type Arabidopsis thaliana Col-0 (purchased from ATCC in USA) by Agrobacterium tumefaciens mediated method (refer to Valvekens et al, 1988) in a medium containing 50. mu.g.L^-1Resistant inbred seedlings were screened on 1/2MS plates of kanamycin and transgenic Arabidopsis T1 inbred plants with MiSKOR overexpression were identified.

Application of a mango root potassium ion channel gene MiSKOR in preparation of a high-efficiency and stress-resistant preparation for improving plant potassium nutrition.

The invention discloses a mango tree K⁺The base sequence of the channel gene MiSKOR is shown as SEQ ID NO: 1 is shown. The gene is mainly expressed specifically in roots, and the expression abundance is highPotassium environment is obviously induced, and mediated K is provided⁺Outward flow and current characteristics of transport.

Secondly, the invention discloses the mango tree K⁺The sequence of the channel protein coded by the channel gene MiSKOR is shown as SEQ ID NO: 2, respectively. Mangifera indica MiSKOR has higher consistency with amino acid sequences of homologous proteins of other plant SKOR, and the amino acid sequences contain functional structural domains such as an ion channel transmembrane domain (PF00520), a cyclic nucleotide binding domain (PF00027), an ankyrin domain (PF12796), and an KHA dimer domain (PF 11834).

Thirdly, the invention provides a recombinant expression vector containing the mango tree MiSKOR gene, wherein the recombinant expression vector is an animal expression vector, and the animal expression vector comprises but is not limited to a vector pTracer-CMV 3.

Fourthly, the invention provides a binary overexpression vector containing the mango tree MiSKOR gene, and the binary expression vector comprises but is not limited to a vector pHB.

Fifth, the present invention provides a gene sequence as set forth in SEQ ID NO: mango tree K shown in 1⁺The application of the channel gene in improving the potassium enrichment and nutrient high-efficiency utilization capacity of crops.

Sixth, the present invention provides a gene sequence as set forth in SEQ ID NO: mango tree K shown in 1⁺Application of channel genes in improving salt stress tolerance of crops.

The MiSKOR gene is cloned from mango for the first time and is subjected to function identification, the MiSKOR gene provided by the invention is mainly specifically expressed at roots, the expression abundance of the MiSKOR gene is obviously induced by a high-potassium environment, and the MiSKOR gene has mediated K⁺The characteristics of outward flowing and transporting current have important application value in the aspects of improving the high-efficiency utilization of potassium nutrition of crops and tolerating salt stress.

Drawings

The invention is further illustrated by the following figures and examples.

FIG. 1 is a diagram of the analysis of the transmembrane structure and the tertiary structure of MiSKOR protein.

Wherein, FIG. 1A is the transmembrane structure of MiSKOR protein; FIG. 1B is the tertiary structure of the MiSKOR protein.

FIG. 2 is a schematic representation of a phylogenetic tree of mango, grape, peach, strawberry, pear, apple, sweet orange, papaya, crimes pomelo, cocoa, cassava, eucalyptus grandis, oilless camphor, poplar, purple willow, cotton, Arabidopsis, tomato, rice and corn SKOR homologous proteins. Drawing notes: mi, mango tree; vvi, grapes; ppe, peach; fve, strawberry; pbr, pear; mdo, apple; csi, sweet orange; cpa, papaya; ccl, criimen pomelo; tca, cacao; mes, cassava; egr, eucalyptus grandis; atr, oil-free camphor; ptr, poplar; spu, purple willow; gra, cotton; ath, arabidopsis; sly, tomato; osa, rice; zma, corn.

FIG. 3 is a schematic representation of the expression profile of the MiSKOR gene.

Wherein A is the expression characteristic of MiSKOR in different tissues or organs, B is MiSKOR to external K⁺A response characteristic of the supply level. Comparison: 1 mmol. L^-1K⁺(ii) a Potassium deficiency: 0 mmol. L^-1K⁺High potassium content: 10 mmol. L^-1K⁺。

FIG. 4 is a schematic diagram of the construction method of pTracer-CMV3-MiSKOR recombinant expression vector.

FIG. 5 shows the restriction enzyme digestion verification of pTracer-CMV3-MiSKOR recombinant expression vector. Drawing notes: m, 1Kbp DNA marker; 1, PCR product of MiSKOR; and 2, enzyme digestion verification of the recombinant plasmid.

FIG. 6 shows K of MiSKOR⁺Schematic representation of the transport characteristics.

FIG. 7 is a schematic representation of potassium enrichment levels in aerial and root sections of the MiSKOR transgenic lines.

FIG. 8 is a schematic diagram of the analysis of salt resistance of MiSKOR transgenic lines.

Detailed Description

Example 1: cloning of mango MiSKOR gene and characteristic analysis of encoded protein

Extracting total RNA of root of Mangifera indica No. 1' by using MiniBEST Plant RNA Extraction Kit (purchased from TaKaRa company), and processing by PrimeScript^TMRT reagent Kit (purchased from TaKaRa company) is reverse transcribed into first strand cDNA, which is used as a PCR template, and a PCR amplification primer is designed by utilizing the full-length CDS electronic sequence of mango MiSKOR gene:

MiSKOR CDS-F:5’-ATGGGCGATACTGCAAGAGAGG-3’(SEQ ID NO：3)

MiSKOR CDS-R:5’-TTACAAGCTCTTCAATGGTATGC-3’(SEQ ID NO：4)

the CDS sequence of MiSKOR obtained by PCR amplification is 2376bp, comprises an initiation codon ATG to a termination codon TAG, codes 791 amino acids, codes the molecular weight of protein of 90.95Kda, has isoelectric point of 7.84, and has 5 typical transmembrane regions. Downloading amino acid sequences of homologous proteins of mango, grape, peach, strawberry, pear, apple, sweet orange, papaya, crime pomelo, cocoa, cassava, eucalyptus grandis, sassafras, poplar, salix purpurea, cotton, arabidopsis thaliana, tomato, rice and corn SKOR in a Phytozome database, and comparing the amino acid sequences of the homologous proteins of 20 plants SKOR by using ClustalX 2.0.13 software to obtain the consistency of 75%. A20-plant SKOR homologous family protein phylogenetic tree is constructed by utilizing an adjacency method in MEGA 7.0, and mango fruit MiSKOR, sweet orange CsiSKOR and ClCitrus cleifra CclSKOR are closely gathered together on the phylogenetic tree.

SEQ ID NO：1：

The coded product is Shaker K⁺Channel protein, amino acid sequence SEQ ID NO: 2:

example 2: expression characteristic analysis of mango MiSKOR gene

RNA of annual leaves, stem segments, roots, young fruits and mature fruits of the 'Tainong No. 1' grafted seedling is extracted, first-chain cDNA is obtained through reverse transcription and is used as a template, a specific expression primer is designed according to the obtained cDNA sequence of MiSKOR, mango MiActin is used as an internal reference, and the tissue specific expression characteristic of the MiSKOR gene is detected through semi-quantitative RT-PCR (the prior art, refer to documents Song and Su, 2013).

Specific primer sequences are as follows:

MiSKOR-F：5’-CAGTGTGAGTGCACTGTTGA-3’(SEQ ID NO：5)，

MiSKOR-R：5’-GAAGGAAAGCCAACTGTCCG-3’(SEQ ID NO：6),

MiActin-F：5’-GAGGAGAAACAGAAGCAAGT-3’(SEQ ID NO：7)，

MiActin-R：5’-AATCATCTTGCTTCTCACCC-3’(SEQ ID NO：8),

RT-PCR results showed that the expression level of the MiSKOR gene was highest in the rootstock and weak in the young fruit, but very low in the leaves and stem. This suggests that the MiSKOR gene may play a role in the potassium nutrition dynamics of mango roots.

Placing the 'Tainong No. 1' grafted seedlings with consistent growth conditions in a medium containing 1 mmol.L^-1KCl was treated with 1/2MS culture medium (formulation references Murashige and Skoog, 1962) for 48 hours, which was a normal control. At the same time, set up lack K⁺And (3) treatment: will 1/2K in MS⁺With Na⁺Instead of K in the culture broth⁺Is 0; high potassium treatment: adding KCl to 1/2MS culture solution to make K⁺Has a final concentration of 10 mmol. L^-1. Then respectively extracting RNA of mango root, stem and leaf tissues under different treatment conditions, carrying out reverse transcription to obtain first-strand cDNA as a template, marking by SYBR Premix ExTaq (TaKaRa) fluorescent dye according to an obtained specific expression primer of MiSKOR and by using mango MiActin as an internal reference, and determining the expression level of MiSKOR gene under different potassium supply conditions by an ABI 7500 real-time fluorescent quantitative PCR instrument.

The real-time fluorescent quantitative PCR result shows that the potassium deficiency obviously inhibits the expression level of the MiSKOR gene in all detected tissues, and the high potassium treatment obviously enhances the expression level of the MiSKOR gene in all detected tissues.

Example 3: cerbera manghas MiSKOR gene K⁺Transport profile analysis

1. Construction of expression vector pTracer-CMV3-MiSKOR

Designing and constructing a specific primer of the recombinant plasmid, wherein a 5' end primer is introduced into a Kpn I restriction enzyme site, and the base sequence is shown as SEQ ID NO: 9(GAGGGTACCA TGGGCGATAC TGCAAGAGAG GA), the 3' end primer introduces Not I enzyme cutting site, and the base sequence is shown as SEQ ID NO: 10(GAAGCGGCCG CTTACAAGCT CTTCAATGGT ATG), the CDS fragment of mango MiSKOR gene cloned in example 1 was amplified by PCR and the PCR product was recovered. PCR recovery products of MiSKOR genes and pTracer-CMV3 plasmids are respectively cut by Kpn I and Not I, then T4 DNA ligase is used for connection, the connection products are transformed into escherichia coli, positive clones are screened, plasmids are extracted by a plasmid minikit, and the recombinant expression vector pTracer-CMV3-MiSKOR is obtained after sequencing verification.

MiSKOR Gene K⁺Transfer characteristics

Preparation of different K⁺Concentration of extracellular fluid: 1.0 mmol. L^-1MgCl 2，1.8mmol·L^-1 CaCl₂， 5.0mmol·L^-1HEPES-NaOH, pH7.4, while ensuring K⁺And Na⁺The total mole number is 100 mmol.L^-1Wherein the final concentration of KCl is 0 mmol.L^-1、10mmol·L^-1And 100 mmol. L^-1. Plasmids of the recombinant expression vector pTracer-CMV3-MiSKOR and the blank vector pTracer-CMV3 were extracted, concentrated and transfected into HEK293-T cells (purchased from Invitrogen), cells with green fluorescence were selected, current signals of channel genes were collected from pCLAMP 10.0(Axon, USA) carried by the patch clamp system (Prior Art, Su et al, 2005; Shenjing et al, 2020; Chen et al, 2021), image collection and analysis of MiSKOR genes at different K by means of pCLAMP 10.0 and SigmaPlut 10.0 software⁺Current versus membrane voltage under extracellular fluid conditions of concentration. Patch clamp voltage application protocol: the clamping voltage is-60 mV, the stepping voltage is 10mV, the total number of 16 stimulation voltages is from-60 mV to +90mV, the stimulation interval is 2s, and the sampling frequency is 10 kHz.

The results are shown in FIG. 6: the HEK293-T cells expressing pTracer-CMV3-MiSKOR recorded a large amount of outward rectifying current, and the outward current intensity was followed by the extracellular K⁺The concentration decreased and the current intensity increased with the increase of membrane voltage, indicating that MiSKOR is a voltage-dependent efflux K⁺A channel.

Example 4: analysis of overexpressed transgenes

1. Expression vector pHB-MiSKOR construction

Designing and constructing a specific primer of the recombinant plasmid, wherein a 5' end primer introduces a BamH I enzyme cutting site, and the base sequence is shown as SEQ ID NO: 11(GAGGGATCCA TGGGCGATAC TGCAAGAGAG GA), XbaI enzyme cutting site is introduced into 3' end primer, and the base sequence is shown as SEQ ID NO: 12(GCGTCTAGAT TACAAGCTCT TCAATGGTAT GC), the CDS fragment of mango MiSKOR gene cloned in example 1 was amplified by PCR and the PCR product was recovered. PCR recovery products of the MiSKOR gene and pHB plasmids are respectively cut by BamH I and XbaI, then T4 DNA ligase is used for connection, the connection products are converted into escherichia coli, positive clones are screened, plasmid is extracted by a plasmid miniprep kit, and the recombinant expression vector pHB-MiSKOR is obtained after sequencing verification.

2. Generation of transgenic Arabidopsis with overexpression

The constructed recombinant expression vector pHB-MiSKOR was infected into the floral foam of wild type Arabidopsis thaliana Col-0 (purchased from ATCC in USA) by Agrobacterium tumefaciens mediated method (refer to Valvekens et al, 1988) in a medium containing 50. mu.g.L^-1Resistant inbred seedlings were screened on 1/2MS plates of kanamycin and transgenic Arabidopsis T1 inbred plants with MiSKOR overexpression were identified.

Sowing MiSKOR overexpression transgenic Arabidopsis T1 generation and wild type Col-0 on 1/2MS solid medium (formula references Murashige and Skoog, 1962), culturing for 1 week, selecting plants with consistent growth, and respectively using seeds containing 1 mmol. L^-1(control) and 10 mmol. L^-1(high potassium) K⁺The potassium content of the whole plant of Arabidopsis thaliana was measured by a spectrophotometer after treatment in 1/2MS nutrient solution at a concentration for one week, and the results are shown in FIG. 7: control (1 mmol. L)^-1K⁺) Under the condition, the potassium content of the MiSKOR over-expression transgenic Arabidopsis T1 and the whole wild Col-0 plant has no obvious difference, and the potassium content is high (10 mmol. L)^-1K⁺) Under the treatment condition, MiSKOR overexpression transformationThe potassium content of the whole gene arabidopsis T1 plant is obviously higher than that of the wild Col-0.

Sowing MiSKOR over-expression transgenic Arabidopsis T1 generation and wild Col-0 on 1/2MS solid medium, culturing for 1 week, selecting plants with consistent growth, and respectively adding seeds containing 0 mmol. L^-1(control) and 100 mmol. L^-1NaCl (salt stress) for one week in 1/2MS nutrient solution, as shown in fig. 8: at 100 mmol. L^-1Under the NaCl treatment condition, the growth of wild Col-0 plants is inhibited, the overground part green losing phenomenon is obvious, the growth condition of MiSKOR overexpression transgenic Arabidopsis T1 is obviously better than that of the wild plants, the overground part biomass and root length are obviously higher than that of the wild plants, and the salt stress tolerance capability is stronger.

The above examples illustrate that the MiSKOR gene resource provided by the present invention is mainly expressed in roots, and the expression level is influenced by the external environment K⁺Horizontal regulation with mediated K⁺Outward flow and transmission function, promoting transgenic plant whole plant K after heterologous overexpression⁺The enrichment capacity of the transgenic plant and the capability of improving the salt stress tolerance of the transgenic plant have important application value in the aspects of improving the high-efficiency utilization and stress resistance of crop potassium.

SEQUENCE LISTING

<110> university of Ludong

<120> mango root potassium ion channel gene MiSKOR, expression vector construction method and application

<130> 111

<140> 202111487516X

<141> 2021-12-07

<160> 12

<170> PatentIn version 3.5

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Trp Ala Leu Tyr Ser Ser Phe Phe Thr Pro Met Glu Phe Ala Phe Phe

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Arg Gly Leu Pro Glu Asn Leu Phe Ile Leu Asp Ile Ala Gly Gln Leu

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Ser Gln Thr Tyr Arg Met Val Tyr Lys His Thr Pro Ile Ala Leu Lys

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Phe Tyr Tyr Leu Ala Thr Thr Leu Pro Pro Gly Glu Glu Gly Tyr Thr

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Trp Ile Gly Ser Leu Lys Leu Gly Asp Tyr Ser Tyr Ser Asn Phe Arg

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Glu Ile Asp Leu Trp Leu Arg Tyr Thr Thr Ser Leu Tyr Phe Ala Ile

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Glu Gly Ala Ser Leu Ser Val Asp Asp Ala Gly Ile Phe Leu Cys Lys

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

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Ala Gly Ala Ser Val Phe Thr Arg Asp Arg Trp Gly Ser Thr Pro Leu

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Met Leu Asp Lys Lys Leu Pro Arg Lys Cys Thr Val Tyr Pro Phe His

755 760 765

Pro Trp Asp Thr Ser Val His Thr Arg His Gly Ile Met Leu Trp Ile

770 775 780

Arg Ile Pro Leu Lys Ser Leu

785 790

<210> 3

<211> 22

<212> DNA

<213> Artificial sequence

<400> 3

atgggcgata ctgcaagaga gg 22

<210> 4

<211> 23

<212> DNA

<213> Artificial sequence

<400> 4

ttacaagctc ttcaatggta tgc 23

<210> 5

<211> 20

<212> DNA

<213> Artificial sequence

<400> 5

cagtgtgagt gcactgttga 20

<210> 6

<211> 20

<212> DNA

<213> Artificial sequence

<400> 6

gaaggaaagc caactgtccg 20

<210> 7

<211> 20

<212> DNA

<213> Artificial sequence

<400> 7

gaggagaaac agaagcaagt 20

<210> 8

<211> 20

<212> DNA

<213> Artificial sequence

<400> 8

aatcatcttg cttctcaccc 20

<210> 9

<211> 32

<212> DNA

<213> Artificial sequence

<400> 9

gagggtacca tgggcgatac tgcaagagag ga 32

<210> 10

<211> 33

<212> DNA

<213> Artificial sequence

<400> 10

gaagcggccg cttacaagct cttcaatggt atg 33

<210> 11

<211> 32

<212> DNA

<213> Artificial sequence

<400> 11

gagggatcca tgggcgatac tgcaagagag ga 32

<210> 12

<211> 32

<212> DNA

<213> Artificial sequence

<400> 12

gcgtctagat tacaagctct tcaatggtat gc 32

Claims (4)

1. A mango root potassium channel gene MiSKOR has a base sequence shown in SEQ ID NO: 1, and the following components:

ATGGGCGATA CTGCAAGAGA GGAAATAGTT GGAGAGGATT TGTCGGATTC TGGCGATTAC

GAAGTAGTAG ATTTGAGAGA TAAGATCAAA TCGTCGCGAG GAAGCCGATT TGATTTAATC

GCCAATCAGT TGCTACGAAG GCGATACTTC AGCCGTCAAA ATGTTATCAC TGGTTTCCAA

GGTCTCCCCA AAACTCTTCT GATCCATCCC GATAACAGTG TGAGTGCACT GTTGACTTTG

TGGAAGAAGT GCAGGTGGTA TCAGGCGTGG ACAAAATTCG TACTTATATG GGCACTCTAC

TCATCCTTCT TTACACCCAT GGAGTTTGCT TTCTTCAGGG GACTTCCAGA GAACCTTTTT

ATTTTGGACA TTGCCGGACA GTTGGCTTTC CTTCTTGATA TTATTTTGCA GTTTTTTCTT

GCCTACAGAG ATAGCCAGAC TTACCGTATG GTTTATAAGC ACACACCCAT TGCTCTCAAG

TACTTAAAAT CCAGTTTTAT CATTGATTTA CTGAGTTGTT TCCCTTGGGA TTTAATTTTT

AAGGCTAGTG GAAGAAGAGA AGAAGTTAGG TATCTGTTAT GGATTAGGTT ATATCGGGTA

CGCAAAGTCA TTCAATTTTT CCATAAGTTG GAGAGGGATA TCCGAATCAA TTATCTGTTT

ACTAGGATTG TGAAACTCAT TGCTGTTGAA CTCTACTGTA CGCACACAGC AGCATGCATC

TTTTACTATT TAGCTACCAC CCTTCCTCCT GGAGAAGAGG GATACACATG GATTGGAAGT

TTAAAATTGG GTGATTATAG CTACTCAAAT TTCAGAGAGA TTGACCTCTG GCTGCGATAC

ACAACATCTT TGTATTTTGC CATTGTAACC ATGGCCACTG TTGGGTATGG GGATATACAT

GCAGTCAATC TGAGGGAAAT GATATTTGTA ATGATTTATG TGTCTTTTGA CATGATTCTT

GGAGCTTACT TAATTGGTAA CATGACAGCA TTAATTGTAA AAGGATCAAA GACGGAAAGG

TTCAGAGATA AAATGGCAGA TGCCATTAAA TACCTGAACA GAAATAGACT TGGGAGGGAC

ATTCGAGACC AGATCAAAGG CCATTTGCGG TTACAGTATG AGAGCAGCTA CACTGAGGCT

GCTGCCCTTC AAGATATCCC TGTTTCCATT CGTGCTAAGA TATCCCAGAC TCTATATTTG

CCGTACATTG AAAAGGTTTA TCTTTTTAAA GGGTGCTCTT CAGAATTTAT CAATCAGATT

GTTATAAGAC TCCATGAGGA ATTTTTTCTC CCTGGAGAAG TGGTGCTGGA GCAGGGAAAT

GTGGTAGATC AACTTTACTT TGTCTGTCAT GGTGTTCTGG AGGAGGTTGG TGTTGGAGAA

GATGGCATGG AAGCTACTGT TTCATATTTA GAACCAAATA GCTCATTTGG AGAGATTTCT

ATTCTTTGCA ACATTCCTCA GCCTTACACT GTTCGGGTTT GTGAACTATG TAGGGTTCTG

CGAATCGATA AACAGTCTTT TGCCAATATC CTTGAGATAT ATTTCTATGA TGGAAGGAAA

ATATTGACCA ACCTCTTAGA GGGTAAGGAT TCCAATATCC GTATTAAACA GCTGGAGTCA

GACATCACAT TTCACATTGC CAAGCATGAA GCAGAACTTG CATTGAGGGT GAATAGTGCA

GCTTATCATG GAGATCTGTA TCAGCTAAAA GGTTTTATTC GGGCTGGAGC AGATCCCAAC

AAGACAGATT ATGATGGGAG GTCACCTTTG CATCTTGCAG CATCAAGAGG ATATGAAGAT

ATTACCATTT TCCTTATACA AGGAGGAGTA GACATTAACA TAAAAGATAA ATTTGGCAAC

ACCCCCTTAC TAGAAGCTTT AAAAAATGGA CATGATCGAG TTTCTTCATT GCTTGCTAAT

GAAGGGGCCT CCTTGAGTGT AGATGATGCT GGTATTTTTC TCTGTAAAGC TGTTGTTAGG

GGGGATACAG ACTTCCTTAA AAGAGTTTTA TCCTATGGTG TTGATCCAAA TTCCAAAGAT

TATGACCACA GAACCCCACT TCATGTAGCT GCCTCAGAGG GACTATATCT GATGGTAAAG

TTGCTTTTAG AGGCTGGGGC TAGTGTTTTT ACAAGGGACA GATGGGGGAG CACCCCTCTT

GATGAAGGTC AGAGGTGTGG AAACAAGAAT TTGATCAAGC TACTGGAAGA AGCAAAATTC

TCTCAGTTGT CAGAATTTCC TCATTGCTCC GAATTCATGC TAGATAAAAA ACTGCCAAGG

AAATGCACTG TGTACCCTTT CCACCCATGG GATACTAGTG TGCATACAAG ACATGGAATT

ATGTTATGGA TTCGCATACC ATTGAAGAGC TTGTAA。

2. a construction method of a recombinant expression vector pTracer-CMV3-MiSKOR of mango root potassium channel gene MiSKOR in claim 1, which is characterized in that: designing a specific primer for constructing a recombinant plasmid, wherein a 5 'end primer is introduced into a Kpn I enzyme cutting site, the base sequence is GAGGGTACCA TGGGCGATAC TGCAAGAGAG GA, a 3' end primer is introduced into a Not I enzyme cutting site, the base sequence is GAAGCGGCCG CTTACAAGCT CTTCAATGGT ATG, amplifying and cloning a CDS fragment of a mango MiSKOR gene by a PCR method, and recovering a PCR product; PCR recovery products of MiSKOR genes and pTracer-CMV3 plasmids are respectively cut by Kpn I and Not I, then T4 DNA ligase is used for connection, the connection products are transformed into escherichia coli, positive clones are screened, plasmids are extracted by a plasmid minikit, and the recombinant expression vector pTracer-CMV3-MiSKOR is obtained after sequencing verification.

3. A method for creating a transgenic arabidopsis thaliana strain with an excessive expression of a mango root potassium channel gene MiSKOR in a heterologous manner, which is characterized in that: comprises the following steps:

(1) expression vector pHB-MiSKOR construction

Designing a specific primer for constructing a recombinant plasmid, wherein a 5 'end primer introduces a BamH I restriction site with a base sequence of GAGGGATCCA TGGGCGATAC TGCAAGAGAG GA, a 3' end primer introduces an XbaI restriction site with a base sequence of GCGTCTAGAT TACAAGCTCT TCAATGGTAT GC, amplifying a Cerbera Manghas MiSKOR gene CDS fragment by a PCR method, and recovering a PCR product; respectively carrying out double digestion on a PCR (polymerase chain reaction) recovery product of the MiSKOR gene and a pHB plasmid by using BamH I and XbaI, then connecting the products by using T4 DNAllagase, transforming escherichia coli by using the connection product, screening positive clones, extracting the plasmid by using a plasmid miniprep kit, and carrying out sequencing verification to obtain a correct recombinant expression vector pHB-MiSKOR;

(2) generation of transgenic Arabidopsis with overexpression

Infecting the constructed recombinant expression vector pHB-MiSKOR into the flower floc of wild type Arabidopsis thaliana Col-0 by the Agrobacterium tumefaciens mediated method, wherein the flower floc contains 50 mu g.L^-1Resistant inbred seedlings were screened on 1/2MS plates of kanamycin and transgenic Arabidopsis T1 inbred plants with MiSKOR overexpression were identified.

4. Application of a mango root potassium ion channel gene MiSKOR in preparation of a high-efficiency and stress-resistant preparation for improving plant potassium nutrition.

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