CN102978216A - Application of OsAKT1 (Oryza sativa L. Arabidopsis K<+> transporter 1) protein in cultivating low-potassium adversity stress-resistant plant - Google Patents

Abstract

The invention discloses an application of an OsAKT1 (Oryza sativa L. Arabidopsis K<+> transporter 1) protein in cultivating a low-potassium adversity stress-resistant plant. The invention provides a method for cultivating the low-potassium adversity stress-resistant plant. The method comprises the following steps: the coding gene of the OsAKT1 protein is transferred into a target plant so as to obtain a transgenic plant with increased low-potassium adversity stress resistant capability; the OsAKT1 protein is obtained from paddy (Oryza sativa) and is the protein of (a) or (b) as follows: (a) the OsAKT1 protein is the protein which consists of an amino acid sequence which is shown in a sequence 1 in a sequence table; and (b) the OsAKT1 protein is the protein which is obtained in the way that the amino acid sequence in the sequence 1 is replaced and/or deleted and/or added with one or more amino acid residues and is derived with the sequence 1 which is related to the low-potassium adversity stress resistance of the plant. The OsAKT1 protein can be used for improving the low-potassium resistant capability of the plants and provides guarantee for cultivating new paddy products which are suitable for potassium deficient soil.

Description

The application of OsAKT1 albumen in cultivating low-kalium resistant environment stress plant

Technical field

The present invention relates to the application of OsAKT1 albumen in cultivating low-kalium resistant environment stress plant.

Background technology

Potassium is the necessary nutritive element of growth and development of plants, is the abundantest monovalent cation of plant materials intensive amount, accounts for the 2%-10% of plant dry weight.Potassium has wide, the characteristics such as content is high, movability is strong that distribute in plant materials, mainly concentrate on the most vigorous position of vital movement, and precedence partition is in the vigorous organ of the vegetative activities such as bud, spire, top stem and the tip of a root and tissue usually.

Potassium does not participate in the formation of any organic substance, but many physiological actions of potassium are that other element institute is irreplaceable.The effect of potassium in plant materials can be divided into following a few class: the activity of regulatory enzyme, absorption and the protein synthesis of promotion nitrogen are regulated the transportation of phloem solute, affect photosynthesis, regulate cell turgor and osmotic potential, keep cell charge balance etc.Thereby the growing of plant, output formation etc. are produced material impact.

Potassium in the soil is the topmost source of potassium of plants element.Under the natural condition potassium element of plant absorbing except small part from the irrigation water, all the other are supplied with by soil entirely, even in the situation of raise crop application of potash fertilizer, the potassium of Crop also has 40-80% from soil.Therefore, the situation of potassium has extremely important meaning to potassium nutrition and the Potassium Fertilizer effect of plant in the soil.Total potassium content of soil generally accounts for the 0.1%-3% of soil gross weight, because plant can only absorb the potassium element that exists with ionic species from soil, therefore wherein only have about 2% potassium directly effective to plant, remaining potassium is not all being fixed in the soil mineral by the form that plant directly absorbs.

The severe situation that at present China's farm crop production development faces is that soil potassium deficiency and Potassic fertilizer resources are most deficient, therefore from considering with long-range strategy economically, understand in depth and be familiar with the reaction mechanism that plant is coerced low potassium, improve the Ability of bearing low potassium of farm crop, significant for the output and the quality that improve farm crop.

Summary of the invention

The purpose of this invention is to provide the application of OsAKT1 albumen in cultivating low-kalium resistant environment stress plant.

The invention provides a kind of method of cultivating the plant of low-kalium resistant environment stress, comprise the steps: the encoding gene of OsAKT1 albumen is imported the purpose plant, obtain the transfer-gen plant that low potassium environment stress tolerance is strengthened;

Described OsAKT1 albumen is available from paddy rice (Oryza sativa), is following (a) or (b):

(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 1;

(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant low-kalium resistant environment stress protein of being derived by sequence 1.

The encoding gene of described OsAKT1 albumen is following 1) or 2) or 3) or 4) dna molecular:

1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 1st to 2805 Nucleotide;

2) dna molecular shown in the sequence 2 in the sequence table;

3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of coding plant low-kalium resistant environment stress associated protein;

4) with 1) or 2) dna molecular with 90% above homology and coded plant low-kalium resistant environment stress associated protein of the dna sequence dna that limits.

Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, hybridizes under 65 ° of C, then uses 2 * SSC, 0.1%SDS and 1 * SSC, 0.1%SDS respectively to wash film once.

The encoding gene of described OsAKT1 albumen can import described purpose plant by expression vector.

Available existing plant expression vector construction contains the recombinant expression vector of described gene.Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, namely comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.When using described gene constructed recombinant plant expression vector, can add any enhancement type promotor or constitutive promoter before its transcription initiation Nucleotide, they can use separately or be combined with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also enhanser be can use, translational enhancer or transcriptional enhancer comprised, but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce enzyme or the gene of luminophor, the antibiotic marker thing with resistance or the anti-chemical reagent marker gene etc. of colour-change as adding the coding that in plant, to express.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Described expression vector specifically can be the multiple clone site of 1301-Ubiq plasmid (between Bam HI and SmaI restriction enzyme site) insert as described in the recombinant plasmid first that obtains of the encoding gene of OsAKT1 albumen.

Carry the expression vector of the encoding gene of described OsAKT1 albumen can be by using conventional biological method transformed plant cells or the tissues such as Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity be led, agriculture bacillus mediated, particle bombardment, pollen tube passage method, and the plant tissue that transforms cultivated into plant.Described gene can import in the described purpose plant by described recombinant plasmid first.

Described purpose plant is monocotyledons or dicotyledons.

Described dicotyledons can be Arabidopis thaliana, such as the environmental Arabidopis thaliana of Colombia or its mutant strain.Described mutant strain specifically can be the atakt1 mutant.

Described " low potassium environment stress tolerance is strengthened " is presented as that plant is to K ⁺Receptivity strengthen and/or plant (underground part of plant and/or over-ground part) in K ⁺Content increases.

The present invention also protects the application of encoding gene in cultivating low-kalium resistant environment stress plant of described OsAKT1 albumen or described OsAKT1 albumen.Described plant is monocotyledons or dicotyledons.Described dicotyledons can be Arabidopis thaliana, such as the environmental Arabidopis thaliana of Colombia or its mutant strain.Described mutant strain specifically can be the atakt1 mutant.

More than arbitrary described " low potassium " to can be potassium concentration be below the 1mM, as below the 500 μ M, below the 250 μ M, below the 100 μ M, below the 50 μ M, below the 5 μ M or 0 μ M.

The present invention also protects encoding gene or the application of above arbitrary described method in plant breeding of described OsAKT1 albumen, described OsAKT1 albumen.Described plant is monocotyledons or dicotyledons.Described dicotyledons can be Arabidopis thaliana, such as the environmental Arabidopis thaliana of Colombia or its mutant strain.Described mutant strain specifically can be the atakt1 mutant.

The present invention also protects the K of described OsAKT1 albumen in participating in organism ⁺Application in the transhipment.

The engineering that the invention discloses rice Os AKT1 albumen and encoding gene thereof is used, disclose especially this gene improve plant to soil in the effectively application aspect the potassium utilising efficiency.OsAKT1 albumen is responsible for regulation and control and absorbs K from soil in paddy rice ⁺The mutant that knocks out of OsAKT1 gene causes paddy rice to K ⁺Absorption significantly reduce, and obvious potassium deficiency foxiness appears.OsAKT1 gene overexpression plant is to K ⁺Receptivity greatly increase.The present invention can be used for improving the plant Ability of bearing low potassium, and the new rice variety that is applicable to the potassium lean soil for cultivation provides guarantee.

Description of drawings

Fig. 1 is the on position of T-DNA in the osakt1 mutant plant.

Fig. 2 is 0.8% agarose gel electrophoresis figure of the pcr amplification product among the embodiment 2.

Fig. 3 is the relative expression quantity of OsAKT1 gene in each strain among the embodiment 2.

Fig. 4 is the result that Southern blot identifies among the embodiment 2.

Fig. 5 is the whole plant photo of rice varieties among the embodiment 2 " Dongjin ", osakt1 mutant.

Fig. 6 is the measurement result of root length, crown length and potassium content among the embodiment 2.

Fig. 7 is the as a result photo of embodiment 3.

Fig. 8 is 0.8% agarose gel electrophoresis figure of the pcr amplification product among the embodiment 4.

Fig. 9 is that the Arabidopis thaliana of each strain among the embodiment 4 is cultivated the photo after 7 days.

Figure 10 is the measurement result of the potassium content of whole plant among the embodiment 4.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples all arranges repeated experiments three times, results averaged.

The osakt1 mutant inserts T-DNA and obtains in the OsAKT1 gene of rice varieties " Dongjin ", available from Rice GE(http: //signal.salk.edu/cgi-bin/RiceGE JOB=TEXT﹠amp; TYPE=GENE﹠amp; QUERY=Os01g45990 is numbered PFG_1B-16021.L; The on position of the T-DNA of osakt1 mutant plant is seen Fig. 1, and wherein solid box is exon, and lines are intron, and ATG is transcripting start point.Can obtain rice varieties " Dongjin " (wild-type plant) from Rice GE equally.

The environmental Arabidopis thaliana (Col-0) of Colombia: TAIR website (http://www.arabidopsis.org/).

Yeast expression carrier p416-GPD(comprises the amino acid selective marker): be purchased from http://www.biomart.cn/infosupply/9602211.htm, article No. is 87360.

Yeast mutation bacterial strain R5421(is known as " strain CY162 " in the literature): reference: Robert L.Nakamura, Julie A.Anderson, Richard F.Gaber. (1997) Determination of Key StructuralRequirements of a K ⁺Channel Pore.THE JOURNAL OF BIOLOGICAL CHEMISTRY272,1011 – 1018.; This sudden change figure bacterial strain is kalium ion transport protein gene mutant.

Wild-type yeast R757(is known as " strain R757 " in the literature): reference: Robert L.Nakamura, Julie A.Anderson, Richard F.Gaber. (1997) Determination of Key Structural Requirementsof a K ⁺Channel Pore.THE JOURNAL OF BIOLOGICAL CHEMISTRY 272,1011 – 1018..

Atakt1 mutant: reference: Xu J, Li HD, Chen LQ, Wang Y, Liu LL, Wu WH. (2006) Aprotein kinase, interacting with two calcineurin B-like proteins, regulates K ⁺TransporterAKT1 in Arabidopsis.Cell, 125:1347-1360; The plant that sets out of this mutant plant is the environmental Arabidopis thaliana of Colombia.

1301-Ubiq plasmid: reference: Baisheng Yu, Zhongwei Lin1, Haixia Li, Xiaojiao Li, Jiayang Li, Yonghong Wang, Xia Zhang, Zuofeng Zhu, Wenxue Zhai, Xiangkun Wang, Daoxin Xie and Chuanqing Sun. (2007) TAC 1, a major quantitative trait locus controllingtiller angle in rice.The Plant Journal 52,891-898; The 1301-Ubiq plasmid is that the ubiquitin promotor is inserted the plasmid that the Pst I restriction enzyme site of PCAMBIA1301 obtains.

Agrobacterium strains GV3101: the reference of mentioning " agrobacterium strains GV3101 ": R.Berres, L.Otten, B.Tinland, E.Malgarini-Clog, B.Walter. (1992) Transformation of vitis tissue by differentstrains of Agrobacterium tumefaciens containing the T-6b gene.Plant Cell Reports 11,192-195..

The acquisition of embodiment 1, OsAKT1 albumen and encoding gene thereof

(http://rice.plantbiology.msu.edu) input LOC_01g45990 obtains the dna sequence dna of one section high affine potassium-channel gene of coding paddy rice in the TIGR website.According to the international plant gene nomenclature of the molecular biology of plants council, the requirement of (Commission for Plant Gene Nomenclature of the International Society forPlant Molecular Biology), with this dna sequence dna called after OsAKT1 gene, coding OsAKT1 albumen.OsAKT1 albumen (is comprised of 935 amino-acid residues) shown in the sequence 1 of sequence table.The open reading frame of OsAKT1 gene is shown in the sequence 2 of sequence table (2808bp).This gene has 11 exons and 10 introns.

The functional verification of embodiment 2, OsAKT1 albumen

Respectively rice varieties " Dongjin " (representing with DJ), osakt1 mutant are carried out respectively following evaluation:

One, Molecular Identification

1, PCR identifies

Extract respectively the underground part (root) of plant seedling and total RNA and the reverse transcription of over-ground part (bizet) and obtain cDNA, take cDNA as template, the primer that forms with Primer1 and Primer2 is to carrying out pcr amplification (target sequence is about 1100bp), to identify the expression amount of OsAKT1 gene.Adopting the OsACTIN gene is house-keeping gene, identifies (target sequence is about 600bp) with the primer that Primer3 and Primer4 form to carrying out PCR.

Primer1：5’-AATGGCTGGTGTCGTAAAGG-3’；

Primer2：5’-AGATGATCGCCGTCTCTGAT-3’。

Primer3：5’-TCCATCTTGGCATCTCTCAG-3’；

Primer4：5’-GTACCCGCATCAGGCATCTG-3’。

0.8% agarose gel electrophoresis figure of pcr amplification product sees Fig. 2.

2, Real-time PCR identifies

Total RNA and the reverse transcription of extracting the plant seedling obtain cDNA, take cDNA as template, adopt the primer of Primer5 and Primer6 composition to carrying out Real-time PCR, to identify the expression amount of OsAKT1 gene.Adopting the OsACTIN gene is house-keeping gene, identifies carrying out Real-time PCR with the primer that Primer7 and Primer8 form.Use SYBR Green Master mix and ABI 7500 sequence detection system (Applied Biosystems) among the Real-time PCR.Comparative CT method is adopted in the processing of data.As internal reference data are carried out normalization method with the OsACTIN gene.

Primer5：5’-TTGCGTTTGAATCGTACAGC-3’；

Primer6：5’-CCTTTACGACACCAGCCATT-3’。

Primer7：5’-TGGTCGTACCACAGGTATTGTGTT-3’；

Primer8：5’-AAGGTCGAGACGAAGGATAGCAT-3’。

As 1, the relative expression quantity of OsAKT1 gene is seen Fig. 3 in the bizet of rice varieties " Dongjin ", the root of osakt1 mutant and the bizet with the expression amount of OsAKT1 gene in the root of rice varieties " Dongjin ".

The result shows: the OsAKT1 gene is normal expression in rice varieties " Dongjin ", and expression amount significantly reduces in the osakt1 mutant; " Dongjin " compares with rice varieties, and the expression amount of OsAKT1 gene has reduced by 98.91% in the osakt1 mutant plant underground part; " Dongjin " compares with rice varieties, and the expression amount of OsAKT1 gene has reduced by 95.73% in the osakt1 mutant above-ground plant parts.

3, osakt1 mutant plant T-DNA inserts the Southern blot evaluation of copy number

Extract the genomic dna of osakt1 mutant plant leaf, carry out 1% sepharose after digesting with different restriction enzymes and carry out electrophoresis, adopt the probe of on-radiation digoxigenin labeled to carry out Southern blot evaluation, the results are shown in Figure 4.The T-DNA fragment of osakt1 mutant is that single copy inserts.

Two, the functional verification of OsAKT1 albumen

1, packet transaction

The compound method of normal water planting liquid: 114.25mg NH ₄NO ₃, 50.38mg NaH ₂PO ₄2H ₂O, 89.25mgK ₂SO ₄, 110.75mg CaCl ₂, 405mg MgSO ₄7H ₂O, 1.88mg MnCl ₂4H ₂O, 92.5 μ g (NH ₄) ₆Mo ₇O ₂₄4H ₂O, 1.17mg H ₃BO ₃, 437 μ g ZnSO ₄7H ₂O, 388 μ g CuSO ₄5H ₂O, 34.75mgFeSO ₄7H ₂O, 46.53mg Na ₂EDTA is settled to 1L with deionized water, and regulating pH with NaOH is 5.70-5.80.

K in the normal water planting liquid ⁺Concentration is about 1mM.

The compound method of low potassium water planting liquid first: change K ₂SO ₄Add-on, other is all with normal water planting liquid.

K in the low potassium water planting liquid first ⁺Concentration is 100 μ M.

The compound method of low potassium water planting liquid second: change K ₂SO ₄Add-on, other is all with normal water planting liquid.

K in the low potassium water planting liquid second ⁺Concentration is 10 μ M.

First group: the rice paddy seed of seed soaking and vernalization is positioned on the filter paper that soaks with deionized water after will sterilizing, and cultivates 7 days in illumination box, then seedling is moved to cultivate after 10 days in the normal water planting liquid to take pictures, measure root cap length and detect potassium content.

Second group: the rice paddy seed of seed soaking and vernalization is positioned on the filter paper that soaks with deionized water after will sterilizing, in illumination box, cultivated 7 days, then seedling is moved to cultivate after 10 days in the low potassium water planting liquid first and take pictures, measure root cap length and detect potassium content.

The 3rd group: the rice paddy seed of seed soaking and vernalization is positioned on the filter paper that soaks with deionized water after will sterilizing, in illumination box, cultivated 7 days, then seedling is moved to cultivate after 10 days in the low potassium water planting liquid second and take pictures, measure root and bizet length and detect potassium content.

2, Phenotypic Observation

The whole plant photo of rice varieties " Dongjin ", osakt1 mutant is seen Fig. 5 A, and equipotential leaf photo is seen Fig. 5 B.Cultivate 10 days in normal water planting liquid after, the whole plant phenotype of rice varieties " Dongjin " and osakt1 mutant is all consistent with leaf morphology, and growth conditions is good.In low potassium water planting liquid, cultivate after 10 days, the osakt1 mutant show leaf chlorosis and occur brown the potassium deficiency spot, and the blade of rice varieties " Dongjin " is still protected and can be kept green, and the root cap height of mutant plant also significantly is lower than rice varieties " Dongjin ".Above result shows that the OsAKT1 gene has participated in low potassium path, is low potassium tolerance gene.

Root and bizet length the results are shown in Figure 6.

3, the mensuration of root cap potassium content

(1) plant is cleaned up with distilled water, underground part (root) and over-ground part (bizet) are separated, be positioned in 80 ℃ of baking ovens and dried four days, to its constant weight.

(2) with underground part and the respectively weighing and record dry weight on analytical balance of over-ground part of oven dry, then put into respectively crucible.

(3) crucible is put into retort furnace, 300 ℃ were toasted 1 hour, then temperature were transferred to 575 ℃ of rebake 7 hours, closed retort furnace, took out crucible after it is cooled to below 200 ℃.

(4) add the 10mL 0.1M HCl aqueous solution in each crucible, with the dissolving residuum.

(5) solution that step (4) is obtained is rear as solution to be measured with the dilution of the 0.1M HCl aqueous solution.

(6) use Z5000 type atomic absorption spectrophotometer planimetry to measure the potassium concn of diluting soln.Adopt KCl production standard curve.

(7) contain how many mg potassium ions in the vegetable material of the every g dry weight of conversion.

Carry out repeated experiments three times, each strain is got 3 strain plant, results averaged in each repeated experiments.

Potassium content the results are shown in Figure 6.

Among Fig. 6: a: root and bizet length (cm); B: root potassium content; C: bizet potassium content.Under the water planting liquid of different concns potassium concentration was cultivated, the potassium content of osakt1 mutant root and bizet all was lower than wild-type, illustrated that the osakt1 mutant is responsive to low potassium, and OsAKT1 is low potassium tolerance gene.After cultivating 10 days under low potassium (the 10 μ M) condition, " Dongjin " compares with rice varieties, and the potassium content of osakt1 mutant plant root has reduced by 42.95%.

Embodiment 3, yeast heterogenous expression OsAKT1 albumen

One, the structure of recombinant expression vector

1, the double chain DNA molecule shown in the sequence 2 of composition sequence table.

2, take the synthetic double chain DNA molecule of step 2 as template, to carrying out pcr amplification, obtain pcr amplification product with the primer of OsAKT1-F and OsAKT1-R composition.

OsAKT1-F：5’-TT GAATTCATGGCGAGGTGGGGCGCCGCT-3’；

OsAKT1-R：5’-TT GTCGACCTAGCTCTTGCCTTTCATCTTCTC-3’。

3, with the pcr amplification product of restriction enzyme EcoRI and SalI double digestion step 2, reclaim enzyme and cut product.

4, with restriction enzyme EcoRI and SalI double digestion Yeast expression carrier p416-GPD, reclaim carrier framework (about 5800bp).

5, the enzyme of step 3 is cut the carrier framework connection that product is connected with step, obtained recombinant plasmid p416-OsAKT1.It is as follows according to sequencing result recombinant plasmid p416-OsAKT1 to be carried out structrual description: inserted the dna molecular shown in the sequence 2 of sequence table between the EcoRI of Yeast expression carrier p416-GPD and SalI restriction enzyme site.

Two, the conversion of yeast

Recombinant plasmid p416-OsAKT1 is imported yeast mutation bacterial strain R5421, obtain yeast transformant R5421-OsAKT1, concrete steps are as follows:

1, picking yeast mutation bacterial strain R5421 mono-clonal is in the YPDA liquid nutrient medium, and 30 ℃, 250r/min are cultured to OD ₆₀₀Be about 1.6-1.8.

2, the bacterium liquid that step 1 is obtained is forwarded to new YPDA liquid nutrient medium by the volume ratio of 1:10, and 30 ℃, 250r/min are cultured to OD600=0.8-1.0, the collecting cell precipitation.

3, step 2 is obtained cell precipitation and be suspended in ddH ₂Among the O, each competent cell is suspended in 60 μ l ddH ₂Then O adds following composition: the PEG4000 aqueous solution of 240 μ l 50g/100mL, the 36 μ L 1.0M LiAc aqueous solution, the SS-DNA (Sigma, D1626) of 10 μ L 5.0mg/mL, 5 μ L recombinant plasmid p416-OsAKT1 (containing 0.1-10 μ g); 30 ℃ of water-baths 30 minutes, then 42 ℃ of water-baths 25 minutes are collected thalline and are coated dull and stereotyped upper 30 ℃ of auxotroph and cultivated 2-3 days.

The preparation method of auxotroph flat board: 6.7g without amino acid yeast nitrogen, 0.77g-Ura DOSupplement, 20g glucose sugar, 15g agar powder with 7.455g KCl is water-soluble and water is settled to 1L, is transferred pH=5.8 with NaOH.

Three, the acquisition of contrast yeast transformant

Replace recombinant plasmid p416-OsAKT1 to carry out step 2 with Yeast expression carrier p416-GPD, obtain yeast transformant R5421-p416-GPD.

Four, yeast complementation experiment

The preparation method of AP solid medium: 20mL 50 * salts solution, 1mL 1000 * trace element solution, 1mL1000 * vitamin solution, 0.77g-Ura DO Supplement, 10mL 100 * Ura mother liquor and 20g glucose are mixed, it is different by add-on to add KCl(, so that the K in the substratum ⁺Concentration is different), be transferred to pH=6.5 with Arginine.

50 * salts solution: contain 400mM H ₃PO ₄, 500mM L-arginine, 100mM MgSO ₄With 10mM CaCl ₂The aqueous solution.

1000 * trace element solution: contain 500 μ g/ml H ₃BO ₄, 50 μ g/ml CuSO ₄, 100 μ g/ml KI, 400 μ g/mlMnSO ₄, 200 μ g/ml Na ₂MoO ₄With 400 μ g/ml ZnSO ₄The aqueous solution.

1000 * vitamin solution: contain 10 μ g/ml biotin(vitamin Hs), 10g/ml nicotinic acid(tobacco acid), 10g/ml inositol(inositol) and 1g/ml pantothenic acid(pantothenic acid) the aqueous solution.

100 * Ura mother liquor: contain 2g/L Ura(uridylic) the aqueous solution.

With wild-type yeast R757(positive control), yeast transformant R5421-OsAKT1(in Fig. 7 with " R5421+OsAKT1 represents "), yeast transformant R5421-p416-GPD(in Fig. 7 with " R5421+p416 " expression) and yeast mutation bacterial strain R5421(negative control) be incubated at AP solid medium (K ⁺Concentration is respectively 50mM, 10mM, 5mM, 1mM, 500 μ M, 250 μ M, 100 μ M or 50 μ M) on, dilute respectively different gradient stigmas and cultivate, observe the growing state of the bacterial plaque of differing materials.

The results are shown in Figure 7.Yeast mutation bacterial strain R5421 and yeast transformant R5421-p416-GPD are at 1mM K ⁺All can not grow below the concentration, and yeast transformant R5421-OsAKT1 is at 50 μ M K ⁺Still can grow during concentration.The result shows, OsAKT1 the is protein mediated K of yeast cell film ⁺Transhipment.

Embodiment 4, transgenic arabidopsis plant Phenotypic examination

One, over-express vector makes up

1, the double chain DNA molecule shown in the sequence 2 of composition sequence table.

2, take the synthetic double chain DNA molecule of step 1 as template, to carrying out pcr amplification, obtain pcr amplification product with the primer of OsAKT1-UN1301F and OsAKT1-UN1301R composition.

OsAKT1-UN1301F：5’–TT AGATCTATGGCGAGGTGGGGCGCCGCT-3’；

OsAKT1-UN1301R：5’–TT CCCGGGCTAGCTCTTGCCTTTCATCTTCTC-3’。

3, with the pcr amplification product of restriction enzyme Bgl II and SmaI double digestion step 2, reclaim enzyme and cut product.

4, with restriction enzyme Bam HI and SmaI double digestion 1301-Ubiq plasmid, reclaim carrier framework (about 11800bp).

5, the enzyme of step 3 is cut the carrier framework connection that product is connected with step, obtained recombinant plasmid.According to sequencing result, it is as follows that the recombinant plasmid first is carried out structrual description: inserted the dna molecular shown in the sequence 2 of sequence table between the Bam of 1301-Ubiq plasmid HI and SmaI restriction enzyme site.

Two, over-express vector arabidopsis thaliana transformation

1, the recombinant plasmid transformed agrobacterium strains GV3101 that step 1 is obtained obtains the Agrobacterium of recombinating.

2, the restructuring Agrobacterium second that step 1 is obtained transforms the atakt1 mutant by the flower infusion method, results T ₁For seed.

3, with T ₁For the plant selfing and gather in the crops seed (T ₂For seed), with T ₂Be T for cultivating seeds ₂For plant.

4, with T ₂Carry out the hygromycin resistance screening for plant at the MS substratum that contains the 50mg/L Totomycin, for a certain T ₁For plant, if its T ₂For the separation ratio of plant demonstration 3:1 in the hygromycin resistance screening, this T ₁Be single copy OsAKT1 gene overexpression plant for plant.

5, will single T that copies OsAKT1 gene overexpression plant ₂For the plant selfing and gather in the crops seed (T ₃For seed), with T ₃Be T for cultivating seeds ₃For plant.

6, with T ₃Carry out the hygromycin resistance screening for plant at the MS substratum that contains the 50mg/L Totomycin, for a certain T ₂For plant, if its T ₃Be the hygromycin resistance plant for plant, this T ₂Be the OsAKT1 gene overexpression plant of isozygotying for plant, this plant and self progeny thereof are an OsAKT1 gene overexpression strain.

Three, turn the acquisition of empty carrier Arabidopis thaliana

Replace the recombinant plasmid first to carry out step 2 with the 1301-Ubiq plasmid, obtain turning the empty carrier plant.

Four, resistance of reverse is identified

Respectively following plant is identified: 2 T that turn OsAKT1 gene Arabidopis thaliana list copy homozygous lines (strain 1, strain 2, strain 3) that choose at random ₃For seed, turn the T of empty carrier plant ₃For the seed of seed, atakt1 mutant and the seed of Colombia's ecotype Arabidopis thaliana.

The Arabidopis thaliana seed is positioned on the MS substratum (the light intensity 60 μ molm of continuous illumination in 22 ℃ of illumination boxs ^-2S ^-1) 4 days, then be divided into two groups, transplant seedlings respectively to MS substratum and low potassium substratum (representing with LK), continuation is cultivated in illumination box after 7 days and is taken pictures, (extract total RNA and reverse transcription is cDNA to Molecular Identification, identify carrying out PCR with the primer that OsAKT1-UN1301F and OsAKT1-UN1301R form) and detect potassium content (whole plant is carried out potassium content measure, method is with 3 of the step 2 of embodiment 2).

MS culture medium prescription: with 1.65g NH ₄NO ₃, 1.9g KNO ₃, 0.17g KH ₂PO ₄, 0.37g MgSO ₄7H ₂O, 0.332g CaCl ₂, 22.3mg MnSO ₄4H ₂O, 8.6mg ZnSO ₄7H ₂O, 0.025mg CoCl ₂6H ₂O, 0.025mgCuSO ₄5H ₂O, 0.025mg Na ₂MoO ₄2H ₂O, 0.83mg KI, 6.2mg H ₃BO ₃, 27.8mg FeSO ₄7H ₂O and 37.3mg Na ₂Water-soluble and the water of EDTA is settled to 1L.

The difference of low potassium substratum and MS substratum is: remove 1.9g KNO ₃, 1.65g NH ₄NO ₃, 0.332gCaCl ₂With 0.17g KH ₂PO ₄, add 706mg Ca (NO ₃) ₂With 144mg NH ₄H ₂PO ₄

PCR evaluation collection of illustrative plates is seen Fig. 8.Photo is seen Fig. 9.Potassium content is seen Figure 10.

In the MS substratum, the phenotype of each strain does not all have significant difference.In the LK substratum, each growth conditions that turns the OsAKT1 gene plant significantly is better than the atakt1 mutant, with Colombia environmental Arabidopis thaliana without significant difference, the phenotype that turns empty carrier Arabidopis thaliana and akt1 mutant strain does not have significant difference.

Claims (10)

1. a method of cultivating the plant of low-kalium resistant environment stress comprises the steps: the encoding gene of OsAKT1 albumen is imported the purpose plant, obtains the transfer-gen plant that low potassium environment stress tolerance is strengthened;

Described OsAKT1 albumen is following (a) or (b):

(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 1;

(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant low-kalium resistant environment stress protein of being derived by sequence 1.

2. the method for claim 1, it is characterized in that: described encoding gene is following 1) or 2) or 3) or 4) dna molecular:

1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 1st to 2805 Nucleotide;

2) dna molecular shown in the sequence 2 in the sequence table;

3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of coding plant low-kalium resistant environment stress associated protein;

4) with 1) or 2) dna molecular with 90% above homology and coded plant low-kalium resistant environment stress associated protein of the dna sequence dna that limits.

3. method as claimed in claim 1 or 2, it is characterized in that: described encoding gene imports described purpose plant by expression vector.

4. such as arbitrary described method in claim 1 or 3, it is characterized in that: described purpose plant is monocotyledons or dicotyledons.

5. method as claimed in claim 4, it is characterized in that: described dicotyledons is Arabidopis thaliana.

6.OsAKT1 albumen or its encoding gene application in cultivating low-kalium resistant environment stress plant;

Described OsAKT1 albumen is following (a) or (b):

(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 1;

(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant low-kalium resistant environment stress protein of being derived by sequence 1.

7. application as claimed in claim 6 is characterized in that: described encoding gene is following 1) or 2) or 3) or 4) dna molecular:

1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 1st to 2805 Nucleotide;

2) dna molecular shown in the sequence 2 in the sequence table;

3) under stringent condition with 1) or 2) the dna sequence dna hybridization that limits and the dna molecular of coding plant low-kalium resistant environment stress associated protein;

4) with 1) or 2) dna molecular with 90% above homology and coded plant low-kalium resistant environment stress associated protein of the dna sequence dna that limits.

8. such as claim 6 or 7 described application, it is characterized in that: described purpose plant is monocotyledons or dicotyledons.

9. the application of arbitrary described method in plant breeding in the claim 1 to 5.

10.OsAKT1 the K of albumen in participating in organism ⁺Application in the transhipment;

Described OsAKT1 albumen is following (a) or (b):

(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 1;

(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant low-kalium resistant environment stress protein of being derived by sequence 1.

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