CN1239705C - Plant low-kalium resistant genes, encoded protein and application thereof - Google Patents

Abstract

The present invention discloses a low kalium resistant gene of a plant, a coded protein thereof and an application thereof. The low kalium resistant gene of a plant of the present invention is one of the following nucleotide sequences: 1) an SEQ ID No. 1 DNA sequence in a sequence list, 2) polynucleotide for coding an SEQ ID No. 2 protein sequence in the sequence list, and 3) a DNA sequence which has more than 90% of homology with a DNA sequence limited by SEQ ID No. 1 in the sequence list and codes the same functional protein. The coded protein of the low kalium resistant gene of a plant is a protein with an SEQ ID No. 2 amino acid residue sequence in the sequence list, or a protein which is used for substituting, deleting or adding one or a plurality of amino acid residues of the SEQ ID No. 2 amino acid residue sequence and has the same activity with the SEQ ID No. 2 amino acid residue sequence and is derived from SEQ ID No. 2. The gene of the present invention can be used for breeding new low kalium resistant plant varieties.

Description

One kind of plant low-kalium resistant gene and proteins encoded and application

Technical field

The present invention relates to a kind of low-kalium resistant gene and proteins encoded and application in the plant biological engineering field, particularly a kind of low-kalium resistant gene and the proteins encoded and the application of Arabidopis thaliana.

Background technology

Potassium plays crucial effects as the essential nutrient element of plant in the g and D of plant, it is many physiology, the biochemical metabolism process of involved in plant not only, also by the function of regulation and control photosynthesis, respiration, material migration and many enzyme systems growth and development of plant, output formation, anti-adversity ability etc. is produced material impact.

At present, along with the minimizing gradually of continuous growth of worldwide population and cultivated area, pressing for people increases grain yield by the mode that improves crop yield.And will address this problem, the situation of the sternness that China faces is that most of arable soil potassium deficiency and potash fertilizer resource are most deficient, this has become the important factor of restriction China farm crop production development.Can improve crop yield significantly although improve the potash fertilizer amount of application, no matter a large amount of import potash fertilizer are from still considering that from long-range strategic importance certain limitation is all arranged economically.

Another approach that solves potash fertilizer shortage in the agriculture production is to improve farm crop from the efficient that absorbs the potassium element under low potassium level.Utilize the means of genetic engineering the gene of regulation and control potassium efficient absorption utilization might to be changed in the farm crop, farm crop can be grown by normal growth in the soil of low potassium, thereby greatly reduce the potash fertilizer demand, reduce production costs.

Arabidopis thaliana is a kind of typical dicotyledonous model plant, and the experimental mouse in its effect and the experimentation on animals, fruit bat, nematode etc. quite have been widely used in plant genetics, developmental biology and molecular biological research.Arabidopis thaliana (Arabidopsis thaliana) is little with its individuality, growth cycle short, genetic background is simply clear, easily oneself becomes the type material of plant science research by characteristics such as mutagenesis.Most of genes of Arabidopis thaliana can both find in other plant, utilize Arabidopis thaliana can be applied to other plant research as the discovery of model plant and help scientist to cultivate new crop varieties, find the method that improves crop yield.

Arabidopis thaliana has about 1.3 hundred million base pairs, 2.9 ten thousand genes.The function of most gene is not clear at present, utilizes mutating technology research gene function to become a kind of effective ways.By research, can obtain the plant function gene that some have significant application value comparatively easily to mutant.

The innovation and creation content

The purpose of this invention is to provide a kind of plant low-kalium resistant gene and a proteins encoded thereof.

Plant low-kalium resistant gene provided by the present invention, name is called AtLKT1 (Arabidopsis thalianaLow-K ⁺Tolerant), deriving from the environmental Arabidopis thaliana of Landsberg, is one of following nucleotide sequences:

1) SEQ ID № in the sequence table: 1 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences.

Dna sequence dna in the sequence table 1 is by 3255 based compositions, and as shown in Figure 1, wherein capitalization is partly represented the opening code-reading frame of the long 1449bp that is made of 15 exons; Lowercase is partly represented the sequence of 14 introns.

The proteins encoded AtLKT1 of plant low-kalium resistant gene A tLKT1, be to have SEQ ID № in the sequence table: the protein of 2 amino acid residue sequences, or with SEQ ID №: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 2 amino acid residue sequence is identical active by SEQ ID №: 2 deutero-protein.

The protein that sequence 2 amino acid residue sequences are made up of 482 amino-acid residues in the sequence table.

Contain expression carrier of the present invention and clone and all belong to protection scope of the present invention.

The primer of arbitrary fragment (bag expand complete sequence) is to also within protection scope of the present invention among the amplification AtLKT1, and wherein, the distance between upstream primer and the downstream primer is between 50 to 5000 bases; The length of each primer of this primer centering is 15 to 30 bases.As, primer 1:AACGCCTTCACGATCGAC (SEQ ID №: 3); Primer 2: CCACCAGTGACGAATTCCA (SEQ ID №: 4)

Utilize certain carrier that can guide foreign gene to express in plant, with AtLKT1 gene transfered plant cell provided by the present invention, or with the AtLKT1 gene overexpression, plant just shows as low-kalium resistant.For the ease of transgenic plant cells or plant being identified and screening, can process employed carrier, as the antibiotic marker thing that adds the alternative mark of plant or have resistance.By the plant transformed host both can be monocotyledons, also can be dicotyledons.It is significant that gene pairs of the present invention is cultivated the low-kalium resistant new variety.

The invention will be further described below in conjunction with drawings and Examples.

Description of drawings

Fig. 1 is the dna sequence dna of AtLKT1

Fig. 2 is the map based cloning (Map-Based Cloning) of AtLKT1

Fig. 3 is that the T-DNA of AtLKT1 gene knockout mutant N536154 inserts the checking result

Fig. 4 is the Southern analytical results that AtLKT1 gene knockout mutant N536154 T-DNA inserts

Fig. 5 is the Northern analytical results of wild-type Ler and mutant lkt1-1

Fig. 6 is the phenotype observations that wild-type and mutant were handled on four kinds of different substratum 4 days

Fig. 7 is the phenotype observations that wild-type and mutant were handled on four kinds of different substratum 10 days

Fig. 8 crosses the expression plant for goal gene and handle 6 days phenotype observations on four kinds of different substratum

Fig. 9 is the pBIB physical map

Embodiment

The map based cloning of embodiment 1, AtLKT1 gene (Map-Based Cloning)

(1) acquisition of the arabidopsis mutant body lkt1-1 of Ler background

Arabidopsis mutant body lkt1-1 is the mutant from the performance low-kalium resistant proterties of row filter acquisition.Wild-type Arabidopis thaliana (the Landsberg ecotype) seed is planted acquisition offspring (M after EMS (Ethyl Methane Sulfonate) mutagenesis ₂Generation), at low potassium (70 μ M-100 μ M K ⁺) screen M on the substratum ₂(annotate: under this low potassium condition, the growth of wild-type plant is suppressed, and typical observability shape or phenotype are that the geotropism growth of seedling root is suppressed fully for seedling.Adopt in the experiment the vertical inverted method of culture dish, the crooked growth of geotropism that therefore shows as root under low potassium condition is suppressed fully); Will be under low potassium condition M ₂For its root in the seedling still can normal growth the strain system of (showing as the crooked growth of geotropism of root) select and plant, obtain offspring (M ₃Generation), to M ₃In generation, detected proterties again under low potassium condition, be to select to carry out genetic analysis and (backcross, obtain F with wild-type with the strain of the above-mentioned low-kalium resistant proterties of stable performance still ₂In generation,, the back was to F ₂The low-kalium resistant proterties in generation detects); Selected material as further gene clone separating than the mutating strain series that meets classical law of inheritance (promptly 1: 3 or 3: 1) of low-kalium resistant in the F2 generation of backcrossing and low-kalium resistant proterties not.Arabidopsis mutant body lkt1-1 of the present invention promptly is by one of low-kalium resistant mutant of aforesaid method screening acquisition.Arabidopis thaliana lkt1-1 mutant show coerce down root at low potassium still can continued growth (showing as the crooked growth of geotropism of root), but and its low-kalium resistant proterties genetic stability; The root growth of wild-type seedling is then suppressed fully under identical low potassium condition.

(2) map based cloning of AtLKT1 gene

At first arabidopsis mutant body lkt1-1 and Col (the environmental Arabidopis thaliana of Columbia) the wild-type hybridization with the Ler background obtains F ₁Generation, F ₁Obtain F for selfing ₂In generation, select the F that on low potassium substratum seedling root still can continued growth (showing as the crooked growth of geotropism of root) ₂Be built into mapping population for individuality.Detailed process is positioned at mutator gene between first chromosomal two marks (nga392 and S0392) with the known molecular mark in the Arabidopis thaliana genetic map as shown in Figure 2; Then with new SSLP and the CAPS mark of TAIR database given information design, with the mutator gene Fine Mapping in the section of the 42kb of BAC clone F12P21.Through the candidate gene in this zone is checked order, determined the sudden change occur in gene A T1G30270 (AT1G30270 for all gene databases relevant with Arabidopis thaliana--as NCBI, GeneBak, the unified numbering of TAIR etc.---middle use is the gene order that analysis is inferred according to base sequence after Arabidopis thaliana whole genome sequence mensuration is finished.Name of AT1G30270 coding is called the albumen of CIPK23, in Arabidopis thaliana, have 20 surplus kind of CIPK, CIPK23 is wherein a kind of.And at present also without any the research report about the CIPK23 function.AtLKT1 gene of the present invention promptly is gene or the CIPK23 gene that is numbered AT1G30270 in the database).The AtLKT1 gene of lkt1-1 mutant takes place one makes the L-Ala (199) in the AtLKT1 albumen become Xie Ansuan by C to the base substitution (1317) of T.The AtLKT1 gene by order-checking lkt1-1 and all candidate genes of Ler in this section obtain (the SEQ ID № in the sequence table: 1), the SEQ ID № in its encoded protein matter such as the sequence table: 2.Among Fig. 2, A is the physical map location of AtLKT1, and the genetic map location is positioned at AtLKT1 in one section zone of BAC clone F12P21; B is the structure of AtLKT1 gene and the mutational site of lkt1-1, and all positions are for initiator codon, and solid frame table shows exon, and line is represented intron between frame.

The T-DNA of embodiment 2, AtLKT1 gene knockout mutant N536154 inserts checking

(1) acquisition of AtLKT1 gene knockout mutant (Salk_036154 or N536154)

Know in the existing Arabidopis thaliana T-DNA insertion of ABRC (the Arabidopsis Biological Resource Center) mutant library have a T-DNA who is numbered Salk_036154 to insert mutant (NASC of this mutant is numbered N536154) according to the message retrieval of TAIR website (http://www.arabidopsis.org), its T-DNA is inserted in the AtLKT1 gene or is numbered the genes encoding zone of ATlG30270.Order the T of this mutant from ABRC ₃For seed, the mutant that AtLKT1 gene that detection and evaluation acquisition are isozygotied or AT1G30270 gene are knocked out.

(2) T-DNA of AtLKT1 gene knockout mutant N536154 inserts checking

Respectively with the seed of wild-type Col, N536154 and lkt1-1 after sterilization, vernalization treatment, on the MS substratum, in the illumination box, carry out continuous illumination and cultivate, treat that growth of seedling moves into MS respectively simultaneously and (contains 20mM K after 4 days ⁺, 3mM Ca ²⁺), low potassium MS (contains K ⁺80 μ M, Ca ²⁺3mM), low calcium MS (contains 20mMK ⁺, 100 μ M Ca ²⁺), and the low calcium MS of low potassium (contain 80 μ M K ⁺, 100 μ M Ca ²⁺) on the solid medium, culture dish is inverted vertically continuation is grown and observe its upgrowth situation continuously in illumination box, 4 kinds of materials (position is shown in Fig. 6 right side) in every ware, every kind of material five strains, the performing PCR of going forward side by side of taking pictures after waiting to grow 4 days detects.Be template with the wild-type Col of CTAB method extraction and the genomic dna of N536154 respectively, carry out pcr amplification according to ordinary method.Then pcr amplification product is detected in 0.8% agarose gel electrophoresis.Result such as Fig. 6 and shown in Figure 3, Fig. 6 show that gene knockout (T-DNA insertion) the mutant plant N536154 of goal gene AtLKT1 (contains K at low potassium ⁺80 μ M) performance and the on all four mutant phenotype of lkt1-1 (Fig. 6 B is seen in the growth bending of main root under the low potassium condition) under the condition; The PCR of Fig. 3 detects and shows that these plant with mutant phenotype all are the individualities that T-DNA isozygotys and inserts.Judge that thus a base mutation that AtLKT1 gene in the lkt1-1 mutant takes place has caused the afunction of AtLKT1 gene.Arrow indication electrophoresis direction among Fig. 3, M is a 2kb dna molecular amount standard; C is the amplified production of Col; Middle 10 swimming lanes insert individual amplified production for N536154 T-DNA isozygotys.

The Southern that embodiment 3, AtLKT1 gene knockout mutant N536154 T-DNA insert analyzes

Extract the genomic dna of wild-type and mutant N536154 with the CTAB method.Get 1 μ g genomic dna, cut with BamHI, EcoRI and HindIII enzyme respectively.Enzyme is cut product and is separated through 0.8% agarose gel electrophoresis (40V), on shaking table, handle running gel with ordinary method then: embathed 15 minutes through 0.25M HCl, sex change liquid (1.5M NaCl, 0.5M NaOH) embathe 30 minutes, neutralizer (1.5M NaCl, 0.5M Tris-HCl, pH 7.2) embathed 30 minutes.Use 10 * SSC that southern blotting technique is arrived HybondN at last ⁺On the nylon membrane.

Probe mark is pressed the Rediprime of Amersham Biosciences ^TMThe explanation of II Random Prime LabellingSystem test kit is carried out.

Prehybridization and hybridization: the Hybond membrane that 2 * SSC prewets is put into hybrid pipe, and the one side that has a dna fragmentation adds 15ml and is preheating to 65 ℃ Church damping fluid and (contains 0.25M Na inwards ₂HPO ₄-NaH ₂PO ₄(pH7.2), 1mM EDTA, 7% (w/v) SDS, 1% (w/v) BSA) 65 ℃ of prehybridizations half an hour approximately, add the good probe of sex change, in 65 ℃ of hybridization 16 hours.

Wash film: after hybridization finishes, use 2 * SSC+0.5% (w/v) SDS respectively, 1 * SSC+0.5% (w/v) SDS, 0.5 * SSC+0.5% (w/v) SDS and 0.1 * SSC+0.1% (w/v) SDS wash film under 65 ℃, each 15 minutes, and constantly use the detector detection signal.

Radioautograph: film washes the back and wraps with preservative film, puts into magazine, sandwiches x mating plate (Kodak product)-80 ℃ of radioautograph in back, develops and photographic fixing after 1-2 days.

The result as shown in Figure 4, the Southern results of hybridization of three kinds of restriction endonucleases shows that all the T-DNA of gene knockout mutant is inserted as single copy.The mutant character that mutant is described is caused by goal gene AtLKT1 sudden change.1,2,3 are respectively the hybridization contrast of Col genomic dna after BamHI, EcoRI and HindIII enzyme are cut among Fig. 4; 4,5,6 is respectively the N536154 genomic dna after BamHI, EcoRI and HindIII enzyme are cut and single band that linear T-dna fragmentation hybridization obtains.

The Northern of embodiment 4, wild-type and mutant lkt1-1 analyzes

In 1/2MS, shake training 8 days Ler and lkt1-1 seedling, move on to low potassium respectively and (contain K ⁺80 μ M, Ca ²⁺3mM) 1/2MS, low calcium (contain K ⁺20mM, Ca ²⁺10 μ M) 1/2MS or low potassium, low calcium (contain K ⁺80 μ M, Ca ²⁺10 μ M) handle in the 1/2MS liquid nutrient medium to take out after 3-12 hour and be used for total RNA and extract; If need root division, hat, then take out the back and cut off from coleoptile, get root, the total RNA of hat material extraction of 100-200mg respectively.The extraction of total RNA is undertaken by the product description of Invitrogen TRIzol Reagent.RNA combines with ultraviolet spectrophotometer and electrophoresis method and to make accurate quantification after 1.0% agarose gel electrophoresis checked for integrity.Carry out following operation then: the denaturing formaldehyde gel of (1) preparation 1.2% (w/v) agarose: 1.2g agarose, 10ml 10 * MOPS damping fluid, 75ml DEPC-H ₂O, heating and melting postcooling add 15ml formaldehyde during to 50-60 ℃; Encapsulating behind the mixing; The gel room temperature is placed after about 1 hour and is put into 1 * MOPS electrophoretic buffer, prerunning 30 minutes (60V) before the last sample, (2) RNA sample preparation: the total RNA of 10-30 μ g adds the RNA loading buffer of 2 times of volumes (v/v), 65 ℃ were heated 5-10 minute behind the mixing, placed 3-5 minute on ice; (3) electrophoresis: in 1 * MOPS electrophoretic buffer, carry out electrophoretic separation; Voltage 10V/cm, about 2cm place stops electrophoresis apart from the edge to bromjophenol blue, needs 2 hours approximately; (4) the gel scanner uni soaks: sweep glue under the ultraviolet lamp, cut unnecessary edge adhesive tape then; Soak twice of gel with flush away formaldehyde with 250ml 10 * SSC, each 20 minutes, soaking finishes can directly change film.(5) change film, prehybridization, hybridization and thereafter step hybridize with Southern.

CDNA full length product with the AtLKT1 gene is a template, and primer sequence is: primer 1:AACGCCTTCACGATCGAC (SEQ ID №: 3); (SEQ ID №: 4), it is that the AtLKT1 gene specific cDNA fragment of 327bp is as probe that pcr amplification goes out length to primer 2: CCACCAGTGACGAATTCCA.The result as shown in Figure 5, A shows inducing that the AtLKT1 expression of gene is subjected to that low potassium coerces, the expression on transcriptional level of mutant lkt1-1 and wild-type does not have difference; B shows that AtLKT1 mainly expresses and is subjected to inducing of low potassium or low calcium in root, mutant is consistent with wild-type on the expression trend of root and hat.Infer that by the Northern results of hybridization phenotypic difference of mutant and wild-type is that difference by the AtLKT1 protein level causes.Among Fig. 5, A is that 8 days seedling of growth moves into different K on the MS substratum ⁺In the 1/2MS solution of concentration, RNA is extracted in sampling behind the processing different time.The denaturing gel electrophoresis collection of illustrative plates of rRNA is as the contrast of mRNA applied sample amount; Hybridization probe is the specific fragment of goal gene AtLKT1; B is that vegetable material is through different K ⁺, Ca ²⁺The 1/2MS solution-treated of concentration is after 12 hours, and root division, hat extract RNA and do hybridization analysis, and four kinds of processing are respectively with 1/2MS solution: MS is for (to contain K ⁺20mM, Ca ²⁺3mM); LK (contains K for low potassium MS ⁺80 μ M, Ca ²⁺3mM); LCa (contains K for low calcium MS ⁺20mM, Ca ²⁺10 μ M); LKLCa (contains K for low potassium, low calcium MS ⁺80 μ M, Ca ²⁺10 μ M).R is a root tissue; S is the over-ground part tissue.

Embodiment 5, wild-type and mutant are handled the phenotype of different time and are observed on four kinds of different substratum

With the seed of two kinds of wild-types (Ler and Col) and mutant lkt1-1, N536154 after sterilization, vernalization treatment, put into illumination box and carry out the continuous illumination cultivation, treat that growth of seedling moves into respectively after 4 days on MS, low potassium MS, low calcium MS and low potassium, the low calcium MS solid medium simultaneously, then culture dish is vertically placed, seedling is inverted continues to put into the illumination box growth and observe its upgrowth situation continuously, 4 kinds of materials (position is shown in Fig. 6 and Fig. 7 right side) in every ware, every kind of material five strains.Handle respectively after 4 days and 10 days and take pictures.

(1) wild-type is observed with the phenotype that mutant was handled on four kinds of different substratum 4 days

Grow and take pictures the result as shown in Figure 6 after 4 days, show: cultivated 4 days 22 ℃ of following continuous illuminations the back of transplanting seedlings, Ler, Col, lkt1-1 and the N536154 plant no significant difference of growing on the MS substratum, root normal growth, generation gravitropism bending (A); On low calcium MS Ler, Col and lkt1-1, N536154 plant root cap still can be grown but the speed of growth is slowed down (C); The growth of Ler and Col obviously is suppressed on low potassium substratum, and growth curvature no longer takes place root system; The root system of lkt1-1 and N536154 plant then continues to take place the crooked growth of gravitropism (B); Then growth further is suppressed on low potassium, low calcium substratum, and the root system of lkt1-1 and N536154 also no longer takes place significantly crooked, near the phenotype (D) of wild-type.Show that when handling 4 days, mutant and wild-type only show evident difference on low potassium substratum, the root system of mutant is coerced energy continued growth down at low potassium, the inhibition that the root growth of wild-type is coerced by low potassium then.Among Fig. 6, A is that MS (contains K ⁺20mM, Ca ²⁺3mM), B (contains K for low potassium MS ⁺80 μ M, Ca ²⁺3mM), C (contains K for low calcium MS ⁺20mM, Ca ²⁺100 μ M), D (contains K for low potassium, low calcium MS ⁺80 μ M, Ca ²⁺100 μ M).

(2) wild-type is observed with the phenotype that mutant was handled on four kinds of different substratum 10 days

Continue to observe Ler, Col, lkt1-1 and the growth traits of N536154 on four kinds of substratum, handles the result that takes pictures after 10 days as shown in Figure 7, show that these four kinds of Arabidopis thaliana plant all can continue quick growth on MS, blade is emerald green, extends hypertrophy; Root system continues elongation (A); On low calcium MS substratum, the blade plumpness of four kinds of Arabidopis thaliana plant is deep green; On low calcium MS substratum one week of growth back four kinds of plant the main root elongation not obvious and replace the lateral root (C) of raised growth; On low potassium MS substratum, the growth of the main root of wild-type Ler and Col continues to be suppressed and only grows the short lateral root of part, leaf growth extends slowly, mutant lkt1-1 corresponding with them and N536154 main root then can continue elongation, cotyledon then shows the chlorosis symptom of potassium deficiency gradually, and this potassium deficiency symptom shows more faster obviously than lkt1-1 in N536154, in the N536154 cotyledon chlorosis not only after 10 days of growth on the low potassium MS, and the part true leaf also begins to occur potassium deficiency chlorosis state (B), infer that this may be different with the genetic background of two kinds of mutant relevant, because the symptom of N536154 corresponding wild type Col growth in low potassium also beginning to show after 10 days cotyledon minus green, then still keep green mostly with lkt1-1 corresponding wild-type Ler cotyledon, that is to say that the Arabidopis thaliana of Col background is more responsive more to low potassium than Ler; Growth is after 10 days in low potassium, low calcium MS substratum, slowly growth and slightly extending, N536154 obviously is suppressed to the growth of two kinds of similar main roots of wild-type of the main root that removes lkt1-1; And four kinds of Arabidopis thalianas all grow some short lateral roots more or less; Leaf growth then shows more obvious chlorosis symptom under low potassium, wherein to the least responsive Ler of low potassium also begin to occur the cotyledon chlorosis to the most responsive N536154 of low potassium then true leaf partly or entirely show chlorosis (D).Among Fig. 7, A is that MS (contains K ⁺20mM, Ca ²⁺3mM), B (contains K for low potassium MS ⁺80 μ M, Ca ²⁺3mM), C (contains K for low calcium MS ⁺20mM, Ca ²⁺100 μ M), D (contains K for low potassium, low calcium MS ⁺80 μ M, Ca ²⁺100 μ M).

The result shows when handling 10 days, mutant and wild-type under low potassium not only root growth show notable difference, and bizet also begins to occur growth differences.The bizet growth differences performance of mutant and wild-type is more obvious on low potassium, low calcium MS substratum.

Embodiment 6, goal gene are crossed and express the phenotype observation that plant was handled 6 days on different substratum

(1) acquisition of the cDNA total length of goal gene AtLKT1

From the Arabidopis thaliana seedling extract total RNA, reverse transcription is cDNA, is template, utilizes a pair of primer be respectively equipped with XbaI and KpnI restriction enzyme site that with this cDNA pcr amplification goes out the goal gene cDNA total length of 1449bp.Primer sequence is as follows:

Primer 1:GCTCTAG ATGGCTTCTCGAACMCGCC (containing the XbaI enzyme cutting site)

Primer 2: ATGGTACC TTATGTCGACTGTTTTGCAATTGTTCC (containing the KpnI restriction enzyme site)

(2) goal gene is crossed and express the phenotype observation that plant was handled 6 days on different substratum

Be connected to (available from Promega company) on the pGEM-T carrier after the cDNA total length recovery with goal gene, connect product transformed into escherichia coli competent cell DH5 α; Transformed bacteria liquid is picking list bacterium colony after kantlex screening, overnight incubation, cuts the positive plasmid of evaluation through PCR and enzyme, further does order-checking and identifies.Select the correct cloned plasmids of sequence to carry out enzyme and cut, with these two kinds of enzymes the plasmid of crossing expression vector pBIB (physical map of this carrier is seen Fig. 9) is carried out enzyme simultaneously and cut with XbaI and KpnI; Disconnected the cutting respectively of the enzyme section of two kinds of plasmids connects with the T4-DNA ligase enzyme after glue reclaims, connect product and transform the competent escherichia coli cell of preparation according to a conventional method again, that the single bacterium colony that transforms out expands is numerous, extract plasmid, is transformed in the competent cell of the agrobacterium tumefaciens of preparation according to a conventional method after PCR, enzyme are cut evaluation and is PCR again and identifies.The positive Agrobacterium bacterium colony that conversion is obtained shakes bacterium through twice and expands numerous back usefulness Floral dip method arabidopsis thaliana transformation.Results obtain T ₁, choose green positive transformed plant and on low potassium substratum, carry out the phenotype observation after hygromycin selection for seed.And then with T ₁Obtain T for plant culture ₂(contain K at four kinds of substratum: A, MS after generation ⁺20mM, Ca ²⁺3mM), B, low potassium MS (contain K ⁺80 μ M, Ca ²⁺3mM), C, low calcium MS (contain K ⁺20mM, Ca ²⁺100 μ M), D, low potassium, low calcium MS (contain K ⁺80 μ M, Ca ²⁺100 μ M) do further checking on: will move on above-mentioned 4 kinds of substratum 6 kinds of materials (position is shown in Fig. 8 right side) in every ware, every kind of material five strains respectively 4 days seedling of growth on the MS substratum.Handle and take pictures after 6 days.The result as shown in Figure 8, show goal gene cross expressed in mutant (N532341) and wild-type (Ler and Col) after, offspring plant equal root crooked property of the similar mutant of phenotype on low potassium substratum; And when handling 6 days, when the blade of contrast N532341 had begun to occur tangible potassium deficiency chlorosis symptom, it is corresponding crossed expression plant and still keeps green.Especially growth after 6 days on low potassium, low calcium substratum, not only three kinds of mistakes leaf look of expressing transformed plant is all near with the leaf form and aspect of wild-type Ler, shows stronger low-kalium resistant characteristic; And N532341 crosses the main root of expressing transformed plant and still can keep normal growth dual the coercing down of low potassium, low calcium.The mistake that shows goal gene is expressed plant and is had stronger low-kalium resistant ability.

Sequence table

<160>4

<210>1

<211>3255

<212>DNA

＜213〉Arabidopsis Arabidopis thaliana (Arabidopsis thaliana)

<400>1

atggcttctc?gaacaacgcc?ttcacgatcg?actccttcac?gatcaacgcc?ttctggtagt 60

tcttctggtg?gtaggacacg?agttggtaag?tatgagcttg?gacgaacttt?gggtgaagga 120

actttcgcta?aggtcaaatt?cgctagaaat?gttgagaatg?gtgataatgt?cgctattaag 180

gttattgata?aagagaaagt?tctgaaaaat?aagatgatcg?ctcaggtatg?tattatacaa 240

ttctccagct?gctgtgttct?tattgggaat?ctgatttacg?cagttgtgat?ataacctttg 300

attttggatt?ttgtagatca?agcgtgagat?ttcgacgatg?aaattgatca?agcacccgaa 360

tgtcattcgt?atgtttgagg?tttgattctg?atactataaa?gctgaattca?aagttgtgtt 420

gcacgttttg?gttgttgttg?ttgttctata?gatttgtgct?ataagctgag?attcgtttga 480

agttatggaa?catatttgtg?gtccctggct?aatcatatgt?atgtaatata?agggatttga 540

ttattctatg?tatttttttg?gtatctttgt?agttacagat?tctcttcttc?ttcttcttct 600

tcttcttctt?cttcttcttc?ttcttcttct?tcttcttctt?cttcttcttc?ttcttcttct 660

tcttcttctt?cttcaggtga?tggctagcaa?aactaagatc?tacttcgttt?tggaattcgt 720

cactggtggg?gagcttttcg?ataaaattgt?aagatttcat?tgaaagatgt?gttttttttt 780

tcttaatact?atgtggaaat?gagatcttaa?ttctttagaa?ttgtcatgta?ttagtcaagc 840

aatgggagat?tgaaggaaga?tgaggcgagg?aagtatttcc?aacagctgat?taatgcggtt 900

gattattgtc?atagcagggg?agtttatcat?agagacctta?aggttaggaa?tcttactttg 960

tgatcctctt?gcatagggtt?tcttgtgaat?caattggcat?cagaataaaa?gtttgatgtg 1020

tttgaatcat?gttgcacatt?ctggttgcag?ccagaaaatt?tgcttttgga?tgctaatggt 1080

gctttgaaag?tgtctgactt?tggattgagc?gctctacctc?agcaagttcg?agtgagcatc 1140

gagatattga?tttctttttc?tatcatttca?ttgtaaatat?catagttcga?taattcagct 1200

actatggctt?ctaaggagaa?agatttccat?gatttattca?aaagctgatt?ttgtgaccct 1260

ttttctttag?gaggatgggt?tacttcacac?aacctgtgga?acacccaatt?atgttgctcc 1320

ggaggtatta?gcaactgcta?ctgaaaactg?acacacaatt?ttctcaaaac?ttgaattcta 1380

ttattttcta?gtctcagtaa?ccttctatat?tttctcccta?ggtaatcaac?aacaaaggtt 1440

atgatggagc?gaaggctgat?ttgtggtctt?gtggagtaat?tctctttgtt?ttaatggctg 1500

gttatttacc?ttttgaagat?tctaacctga?cgtcgttata?taaaaaggta?ttacgcttgc 1560

ctcttcttct?tttgcttttc?ttttgataca?tatcatttta?catgctatca?gaaaggcttc 1620

aaggtctagg?tgcatttggg?tggctgtata?aaagcctctg?tcatttttgt?atgagacgta 1680

tatagcataa?aagccacctg?attttctttc?ttcataatta?cagatattca?aagcagaatt 1740

tacttgccct?ccctggttct?ctgcaagtgc?taagaagtta?atcaaaagaa?ttctggatcc 1800

caatccagca?acggtatatt?tttcttaccc?tattatataa?tttatattag?aggatctctc 1860

ctttgaagag?gcaaaagctt?ctataggatt?gagtagatct?gaaggcttac?actgattttt 1920

tatattgggc?agaggattac?atttgctgag?gtcattgaaa?atgagtggtt?taaaaaaggg 1980

tataaagcac?ctaaatttga?gaatgctgat?gtcagccttg?atgatgttga?tgcaatcttt 2040

gatgactcag?gggtaaggct?agtgttcttt?tgtttgtttt?tttcggtact?ttgtccggga 2100

tgattgctca?gttgtttttg?tttgttaata?ggagtctaag?aatcttgttg?tggagaggcg 2160

agaagaagga?ctcaaaacac?cagtaacgat?gaatgctttt?gagctcatct?cgacatccca 2220

gggtctcaat?ctcggttcac?ttttcgaaaa?acaaatggta?cagaagtctg?atacgtaaag 2280

ctggtcttta?aatttcgtcc?cccaaggctc?ttattttgtt?gacaattttt?aactatcttt 2340

ttgcggtttt?ggtcaaacct?aataagaagt?agctttgcca?caaaatttta?gtgaccaata 2400

cttttctttg?gaagcccgta?cttagtttga?tagaagtcgt?cattctgatt?gataagcttc 2460

tctgatttca?ggggctggtg?aaacgaaaaa?ctcgatttac?ttccaaatct?tcggctaatg 2520

agatagttac?aaaaattgag?gctgcggcag?cacctatggg?gtttgatgtc?aagacaaata 2580

actacaaggt?tagaatactg?ttacgtattt?acccaatgct?gtggagttgt?gtattggttt 2640

gttgcttctg?aatagaccct?ttaaccattt?ctcaactcct?tcagatgaag?ctgacaggag 2700

agaaatcagg?ccgcaagggt?cagttggcag?ttgccactga?ggttagcttt?cacaatgacc 2760

agtgatcacc?ctttgtatgg?ttggctgaac?ctaatttaag?ttaattgaca?tctaccttca 2820

ggtttttcaa?gttgctccat?ctctttacat?ggttgaaatg?cgaaaatcag?ggggtgacac 2880

attggaattc?cacaaggtat?gcaagggtta?ttaatgctga?tcaggttgta?tcaaacccga 2940

gcttgtcaaa?tggtcttcca?gtaactgatg?tttgaatctt?tacctttttg?catcctttct 3000

tgctcttctt?ttagttttac?aagaacctta?ccacgggact?taaggacatt?gtttggaaaa 3060

ccatcgacga?agagaaagag?gaaggaaccg?atggtggtaa?gtatctgcct?tgaatactct 3120

gttttttaga?taaagctcct?agaaaaggag?aaactaacga?aacatgtaaa?cacaaatcct 3180

gttttgtttc?ttcatgtttc?aggtggtact?aatggtgcca?tggctaaccg?gacaattgca 3240

aaacagtcga?cataa 3255

<210>2

<211>482

<212>PRT

＜213〉Arabidopsis Arabidopis thaliana (Arabidopsis thaliana)

<400>2

Met?Ala?Ser?Arg?Thr?Thr?Pro?Ser?Arg?Ser?Thr?Pro?Ser?Arg?Ser?Thr

1 5 10 15

Pro?Ser?Gly?Ser?Ser?Ser?Gly?Gly?Arg?Thr?Arg?Val?Gly?Lys?Tyr?Glu

20 25 30

Leu?Gly?Arg?Thr?Leu?Gly?Glu?Gly?Thr?Phe?Ala?Lys?Val?Lys?Phe?Ala

35 40 45

Arg?Asn?Val?Glu?Asn?Gly?Asp?Asn?Val?Ala?Ile?Lys?Val?Ile?Asp?Lys

50 55 60

Glu?Lys?Val?Leu?Lys?Asn?Lys?Met?Ile?Ala?Gln?Ile?Lys?Arg?Glu?Ile

65 70 75 80

Ser?Thr?Met?Lys?Leu?Ile?Lys?His?Pro?Asn?Val?Ile?Arg?Met?Phe?Glu

85 90 95

Val?Met?Ala?Ser?Lys?Thr?Lys?Ile?Tyr?Phe?Val?Leu?Glu?Phe?Val?Thr

100 105 110

Gly?Gly?Glu?Leu?Phe?Asp?Lys?Ile?Ser?Ser?Asn?Gly?Arg?Leu?Lys?Glu

115 120 125

Asp?Glu?Ala?Arg?Lys?Tyr?Phe?Gln?Gln?Leu?Ile?Asn?Ala?Val?Asp?Tyr

130 135 140

Cys?His?Ser?Arg?Gly?Val?Tyr?His?Arg?Asp?Leu?Lys?Pro?Glu?Asn?Leu

145 150 155 160

Leu?Leu?Asp?Ala?Asn?Gly?Ala?Leu?Lys?Val?Ser?Asp?Phe?Gly?Leu?Ser

165 170 175

Ala?Leu?Pro?Gln?Gln?Val?Arg?Glu?Asp?Gly?Leu?Leu?His?Thr?Thr?Cys

180 185 190

Gly?Thr?Pro?Asn?Tyr?Val?Ala?Pro?Glu?Val?Ile?Asn?Asn?Lys?Gly?Tyr

195 200 205

Asp?Gly?Ala?Lys?Ala?Asp?Leu?Trp?Ser?Cys?Gly?Val?Ile?Leu?Phe?Val

210 215 220

Leu?Met?Ala?Gly?Tyr?Leu?Pro?Phe?Glu?Asp?Ser?Asn?Leu?Thr?Ser?Leu

225 230 235 240

Tyr?Lys?Lys?Ile?Phe?Lys?Ala?Glu?Phe?Thr?Cys?Pro?Pro?Trp?Phe?Ser

245 250 255

Ala?Ser?Ala?Lys?Lys?Leu?Ile?Lys?Arg?Ile?Leu?Asp?Pro?Asn?Pro?Ala

260 265 270

Thr?Arg?Ile?Thr?Phe?Ala?Glu?Val?Ile?Glu?Asn?Glu?Trp?Phe?Lys?Lys

275 280 285

Gly?Tyr?Lys?Ala?Pro?Lys?Phe?Glu?Asn?Ala?Asp?Val?Ser?Leu?Asp?Asp

290 295 300

Val?Asp?Ala?Ile?Phe?Asp?Asp?Ser?Gly?Glu?Ser?Lys?Asn?Leu?Val?Val

305 310 315 320

Glu?Arg?Arg?Glu?Glu?Gly?Leu?Lys?Thr?Pro?Val?Thr?Met?Asn?Ala?Phe

325 330 335

Glu?Leu?Ile?Ser?Thr?Ser?Gln?Gly?Leu?Asn?Leu?Gly?Ser?Leu?Phe?Glu

340 345 350

Lys?Gln?Met?Gly?Leu?Val?Lys?Arg?Lys?Thr?Arg?Phe?Thr?Ser?Lys?Ser

355 360 365

Ser?Ala?Asn?Glu?Ile?Val?Thr?Lys?Ile?Glu?Ala?Ala?Ala?Ala?Pro?Met

370 375 380

Gly?Phe?Asp?Val?Lys?Thr?Asn?Asn?Tyr?Lys?Met?Lys?Leu?Thr?Gly?Glu

385 390 395 400

Lys?Ser?Gly?Arg?Lys?Gly?Gln?Leu?Ala?Val?Ala?Thr?Glu?Val?Phe?Gln

405 410 415

Val?Ala?Pro?Ser?Leu?Tyr?Met?Val?Glu?Met?Arg?Lys?Ser?Gly?Gly?Asp

420 425 430

Thr?Leu?Glu?Phe?His?Lys?Phe?Tyr?Lys?Asn?Leu?Thr?Thr?Gly?Leu?Lys

435 440 445

Asp?Ile?Val?Trp?Lys?Thr?Ile?Asp?Glu?Glu?Lys?Glu?Glu?Gly?Thr?Asp

450 455 460

Gly?Gly?Gly?Thr?Asn?Gly?Ala?Met?Ala?Asn?Arg?Thr?Ile?Ala?Lys?Gln

465 470 475 480

Ser?Thr

<210>3

<211>18

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>3

aacgccttca?cgatcgac 18

<210>4

<211>19

<212>DNA

＜213〉artificial sequence

<220>

<223>

<400>4

ccaccagtga?cgaattcca 19

Claims (6)

1, a kind of plant low-kalium resistant gene is one of following nucleotide sequences:

1) SEQ ID № in the sequence table: 1 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences.

2, the proteins encoded of a kind of plant low-kalium resistant gene, be to have SEQ ID № in the sequence table: the protein of 2 amino acid residue sequences, or with SEQ ID №: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 2 amino acid residue sequence is identical active by SEQ ID №: 2 deutero-protein.

3, contain the described expression carrier of claim 1.

4, the transgenic cell line that contains the described gene of claim 1.

5, the primer of the described gene complete sequence of amplification claim 1 is right.

6, the application of the described gene of claim 1 in cultivating the low-kalium resistant plant variety.

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