CN103012574A - Low-phosphor stress response regulatory factor ZmPHR1, gene for coding the protein and application - Google Patents

Abstract

The invention discloses a low-phosphor stress response regulatory factor ZmPHR1, a gene for coding the protein and an application thereof, belonging to the technical field of genetic engineering and aiming at solve a problem that the phosphor utilization rate of the crops is low by a molecular breeding method. The amino acid sequence of the low-phosphor stress response regulatory factor ZmPHR1 is shown as SEQ ID NO: 2; or the sequence is replaced, deleted or added with one or more amino acid sequences, with the same function, of the amino acid. The invention further provides a gene for coding the regulatory factor ZmPHR1, wherein the sequence of the gene contains the nucleotide sequence shown as the SEQ ID NO: 1; and the invention discloses the application of the gene in plant breeding. The gene and the coding protein of the low-phosphor stress response regulatory factor ZmPHR1 provided by the invention have an important action in genetic improvement of the grains such as corns, wheat, rice and the like, and the industrial crops, and have an extensive application prospect.

Description

Gene and the application of a kind of low phosphorus response regulatory factor ZmPHR1, this albumen of encoding

Technical field

The invention belongs to gene engineering technology field, relate to gene and the application of a kind of low phosphorus response regulatory factor ZmPHR1, this albumen of encoding.

Background technology

The shortage of Phosphorus in Soil is a global problem, and the concentration of phosphorus only has 1～10 μ M in the arable land of 30-40%, and so low phosphorus content can not satisfy the needs of plant-growth and growth.Usually people guarantee the normal growth of plant and obtain higher crop yield by using phosphate fertilizer, yet, even sufficient phosphate fertilizer supply is arranged, most of phosphate fertilizer also can with alkaline soil in calcium ion or the aluminum ion in the acid soil and iron ion form insoluble compound, perhaps form organic substance with microbial process, only have the phosphorus element about 20% to be utilized by plant absorbing; In addition, the part inorganic phosphorus in the soil enters in the underground water by rain-out or runoff, causes soil body eutrophication, and this situation more and more becomes the environmental problem of a globalization.In addition, the too much inorganic phosphorus fertilizer of using also is a kind of consumption and waste to non-renewable phosphate rock resource all the year round, finally causes having improved the volume cost of crop.

In order to solve the low problem of crop phosphorus utilization, be necessary the phosphate use regulatory mechanism that lacks under the phosphorus condition is carried out awareness and understanding, thereby adopt molecular breeding method to improve this proterties of crop.

Under scarce phosphorus condition, plant has formed a series of adaptation mechanism.Have been reported, in Arabidopis thaliana, have 29% transcription factor relevant with the phosphate starvation reaction.Transcription factor such as WRKY75, ZAT6, MYB62, BHLH32, AtPHR1 and PHR1-LIKE1 are the main regulators of regulation and control phosphorus element running balance in the different plants.Wherein, the AtPHR1 that belongs to MYB-CC family a member be in the Arabidopis thaliana first transcriptional level be proved to be to phosphate starvation rise regulating and controlling effect transcription factor it with one section incomplete palindromic sequence (P1BS of dimeric form and a series of phosphate starvation responsive genes promoter regions, GNATATNC) combination, thereby expression (the Rubio et al. 2001 of regulation and control corresponding gene; Franco-Zorrillaet al. 2007; Nilsson et al. 2007).AtPHR1 is carried out transgenic research, and the result shows, the content of inorganic phosphorus (the Bari et al. 2006 that all is significantly improved in the wild-type strain after conversion and the mutant strain; Nilsson et al. 2007), illustrate that AtPHR1 plays a role aspect the utilizing of phosphorus improving.In recent years, isolated OsPHR2 and AtPHR1 are a bit similar from paddy rice, in the strain of overexpression OsPHR2, the expression of several phosphate transporter genes has all obtained rise, think with stem in the raising of phosphorus content be proportionate, thereby infer that OsPHR2 is also playing regulating effect (Zhou et al. 2008) aspect the rice phosphorus utilising efficiency improving.

Corn is in the world the most important cultivated plant and cash crop, major part all grows in acidity and alkaline soil, because phosphate fertilizer is easy to be adsorbed by soil particle, causes forming the phosphorus compound of insolubility, has influence on the corn normal growth and obtain high yield.There are some researches show that there is genotypic difference in the different germplasm materials of corn to the utilising efficiency of phosphorus, by the phosphate use efficient screening study of greenhouse and field test, only has few part self-mating system to be considered to belong to phosphorus efficiency and utilizes genotype.And the efficient utilization of phosphorus belongs to quantitative character, be subjected to controlled by multiple genes, the importing of transcription factor can be regulated and control the expression of downstream a series of phosphate use correlation function gene, be a kind of practicable method (Century et al. 2008) to improving crop phosphate use efficient.Yet, up to the present, in corn, the report of the transcription factor similar to PHR1 is arranged not yet.

The present invention studies have shown that by separation, sequential analysis and the allos functional verification etc. to Maize Transcription Factor ZmPHR1, overexpression ZmPHR1 in Arabidopis thaliana, regulated and control the expression of a series of phosphorus induced genes and phosphate transporter gene, thereby the susceptibility that plant is coerced phosphorus has obtained alleviation, improve the content of phosphorus in the growing way of plant under the low-phosphorous condition and the cauline leaf, thereby provide basic substance for the breeder improves phosphate use efficient by the genetic engineering means.

Summary of the invention

In order to adopt molecular breeding method to improve the low problem of crop phosphorus utilization, the invention provides gene and the application of a kind of low phosphorus response regulatory factor ZmPHR1, this albumen of encoding.

The present invention adopts following technique means to realize:

A kind of corn low phosphorus response regulatory factor ZmPHR1, its aminoacid sequence are shown in SEQ ID NO:2, or this sequence is through replacing, lack or adding the formed aminoacid sequence with same function of one or several amino acid.

The present invention also provides the gene of the above-mentioned regulatory factor ZmPHR1 that encodes, and the sequence of described gene contains the nucleotide sequence shown in the SEQ ID NO:1.

The present invention also provides the plant expression vector that contains said gene.

Described plant expression vector comprises:

(1) plant expression vector is pJIT163-ZmPHR1 ∷ GFP, and the various intermediate carriers that generate in building process, is described gene to be inserted between the Sal I of pJIT163 ∷ GFP and the BamH I restriction enzyme site obtain;

(2) plant expression vector is pCAMBIA1300-ZmPHR1 ∷ GFP, and the various intermediate carriers that in building process, generate, be that the small segment that restriction enzyme KpnI is connected with Xho I double digestion recombinant plasmid first is obtained with the large fragment connection that restriction enzyme KpnI is connected with SalI double digestion pCAMBIA1300.

The present invention also provides a kind of agrobacterium strains, is that the recombinant plasmid that will contain plant expression vector pCAMBIA1300-ZmPHR1 ∷ GFP imports agrobacterium strains GV3101 acquisition by freeze-thaw method.

The present invention also provides the transgenosis homozygous lines of overexpression, is to import in the Arabidopis thaliana by the Agrobacterium bacterium liquid topical application that will contain plant expression vector pCAMBIA1300-ZmPHR1 ∷ GFP to obtain.

The increase primer pair of the total length of described gene or its any fragment of the present invention, described primer sequence is as follows:

Primer is to 1

Forward primer: 5 '-AAGCAAGGTAATGAAATG-3 ';

Reverse primer: 5 '-TGAATAGTGCAACCGATA-3 '.

Primer is to 2

Forward primer: 5 '-CACCCTTTATTTCTCAGTCATCCAA-3 '

Reverse primer: 5 '-TCATTTTGTGTAGCACTCTCATCAG-3 '

The present invention also further provides the application of said gene in plant breeding.

Under low-phosphorous condition, the phosphorus content utmost point that turns ZmPHR1 gene strain is significantly higher than the wild-type plant, ZmPHR1 has increased the absorption of transfer-gen plant to phosphorus after crossing expression, thereby can improve the P Nutrient of plant, so that the plant height of plant, biomass is compared all with the wild-type plant obviously to be increased, and root/shoot ratio is compared all with the wild-type plant and obviously reduced, as shown in Figure 5.Owing to cross and to express the suction phosphorus ability that ZmPHR1 can increase plant, thereby under the condition of same soil fertility, particularly under the low-phosphorous condition, the ZmPHR1 transfer-gen plant is can Shaoshi fertile, reduces Soil Environmental Pollution, economizes on resources.Gene of the present invention and proteins encoded thereof particularly will play an important role in the breed improvement of the grain such as corn, wheat and paddy rice and cash crop in crop, have a extensive future.

Description of drawings

The structural representation of Fig. 1 pJIT163-ZmPHR1 ∷ GFP;

The structural representation of Fig. 2 pCAMBIA1300-ZmPHR1 ∷ GFP;

Fig. 3 transgenic arabidopsis homozygous lines;

Fig. 4 transgenic line PCR detected result;

Fig. 5 transgenic line RT-PCR detected result;

Fig. 6 transgenic arabidopsis plant GFP fluorescence display result;

GFP fluorescence display result in Fig. 7 transgenic arabidopsis plant root;

The acquisition of Fig. 8 overexpression strain;

Fig. 9 ZmPHR1 overexpression strain and the wild-type strain growing state under high phosphorus (1mM Pi) and low-phosphorous (10 μ M Pi) condition;

Figure 10 ZmPHR1 overexpression strain and wild-type strain, the phosphorus content of plant root and overground part under high phosphorus (1mM Pi) and low-phosphorous (0.01mM Pi) condition;

Figure 11 ZmPHR1 overexpression strain and wild-type strain, the expression of phosphorus responsive genes under high phosphorus (1mM Pi) and low-phosphorous (0.01mM Pi) condition;

Figure 12 ZmPHR1 overexpression strain and wild-type strain, the expression of phosphate transporter gene under high phosphorus (1mM Pi) and low-phosphorous (0.01mM Pi) condition.

Embodiment

Experimental technique among the following embodiment, material if no special instructions, is ordinary method and purchases availablely from routine biochemistry reagent shop, and % wherein if no special instructions, is the quality percentage composition.

Quantitative test in following examples all arranges repeated experiments three times, results averaged.

The T1 representative shows that T0 reaches the plant that is grown up to by it for the seed that selfing produces, and the T2 representative is shown T1 for the seed of selfing generation and the plant that is grown up to by it, and the T3 representative shows that T2 is for the seed of selfing generation and the plant that is grown up to by it.

Corn material 478: for Shanxi Agricultural academy of sciences modern agriculture research centre breeding chamber is preserved.

The pJIT163-GFP carrier: Inst. of Genetics and Development Biology, CAS gives.

Plasmid pCAMBIA1300: give from Chinese Academy of Sciences heredity and developmental biology institute.

Agrobacterium GV3101: give from Chinese Academy of Sciences heredity and developmental biology institute.

The environmental Arabidopis thaliana of Colombia: give from Chinese Academy of Sciences's heredity and developmental biology institute.

Implement discovery and the clone of 1 ZmPHR1 albumen and encoding gene thereof

Aminoacid sequence according to the AtPHR1 (At4g28610) of Arabidopis thaliana, search TIGR (The Institute of Genomic Research Database-TDB) corn database (http://maize.jcvi.org), length and structural domain according to homology, predicted amino acid sequence, may the encode collating sequence TC346249 of PHR1 gene of acquisition amounts to 13 corn EST.

Take TC346249 as template, design PCR primer sequence is as follows:

Forward primer: 5 '-AAGCAAGGTAATGAAATG-3 ';

Reverse primer: 5 '-TGAATAGTGCAACCGATA-3 '.

Extract total RNA of self-bred line 478 with the Trizol reagent of Invitrogen company, after DNaseI (RNase-free) processes, get 4 μ g and carry out synthetic cDNA the first chain of reverse transcription with the M-MLV ThermoScript II of Promega company, carry out the PCR amplification with it as template

The PCR reaction system: ultrapure water 39.5 μ L, 10 * PCR buffer, 5 μ L, template 2 μ L, concentration is each 1 μ L of forward and reverse primer of 10 μ M, EX-TaqDNA enzyme (5u/ μ L) 0.5 μ L, dNTPs (10mM) 1 μ L.

PCR reaction conditions: 94 ℃ of 4min; 94 ℃ of 30sec, 53/60 ℃ of 30sec, 72 ℃ of 1min30sec, 35 circulations; 72 ℃ of 10min; 4 ℃ of insulations.

Obtain the PCR amplified production, with reference to Beijing ancient cooking vessel state biotechnology limited liability company product dna fragment fast purifying/recovery test kit purifying PCR amplified production, spend the night under 16 ℃ with the pMD18-T carrier and to be connected, obtain recombinant plasmid pMD18-ZmPHR1, use the thermal shock method that recombinant plasmid pMD18-ZmPHR1 is transformed bacillus coli DH 5 alpha, conversion product is grown at the LB plate culture medium that contains penbritin, selects positive colony.From positive colony, extract plasmid, check order.

Obtain 1 complete ORF after the order-checking, namely SEQ ID NO:2 aminoacid sequence in the sequence table is inferred the polypeptide that is comprised of 449 amino-acid residues, and this albumen called after ZmPHR1 contains typical MYB and Coiled-coil structural domain.With the encoding gene called after ZmPHR1 gene of ZmPHR1 albumen, its open reading frame nucleotide sequence is 1350bp (containing terminator codon) shown in SEQ ID NO:1.

Implement the structure of 2 plant expression vectors

1.pJIT163-ZmPHR1 the structure of ∷ GFP expression vector

Concrete steps are as follows:

(1) introduces SalI and BclI site (BclI is the isocaudarner of BamH I) at the gene two ends of recombinant plasmid pMD18-ZmPHR1

The PCR primer:

Forward primer: 5 '-GC gtcgac ATGAGGAAGTTTAATC-3 '

Reverse primer: 5 '-GCG tgatca ACTATCTTGCAGTTT-3 '

The PCR reaction system: ultrapure water 39.5 μ L, 10 * PCR buffer, 5 μ L, template 2 μ L, concentration is each 1 μ L of forward and reverse primer of 10 μ M, EX-TaqDNA enzyme (5u/ μ L) 0.5 μ L, dNTPs (10mM) 1 μ L.

PCR reaction conditions: 94 ℃ of 4min; 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 1min30sec, 35 circulations; 72 ℃ of 10min; 4 ℃ of insulations.

(2) recombinant plasmid pMD18-ZmPHR1 is carried out double digestion with restriction enzyme SalI and BclI, reclaim the gene fragment with restriction enzyme site.

(3) with restriction enzyme Sal I and BamH I double digestion recombinant plasmid pJIT163-GFP, reclaim carrier framework.

(4) fragment that step (2) is reclaimed is connected 3 with step) carrier framework that reclaims connects, and obtains recombinant plasmid pJIT163-ZmPHR1 ∷ GFP; The SEQ ID NO:1 of sequence table inserts between the two 35S promotors and SalI and BamH I restriction enzyme site between the GFP gene of pJIT163-GFP from the ZmPHR1 gene shown in 5 ' terminal the 1st to 1347 Nucleotide, sees Fig. 1.

2.pCAMBIA1300-ZmPHR1 the structure of ∷ GFP expression vector

Concrete steps are as follows:

(1) with restriction enzyme KpnI and Xho I double digestion recombinant plasmid first (pJIT163-ZmPHR1 ∷ GFP), reclaims the fragment (containing 35S promotor, ZmPHR1 gene, GFP gene and terminator) about about 3.6kb.

(2) with restriction enzyme KpnI and Sal I double digestion plasmid pCMBIA1300, reclaim carrier framework.

(3) fragment that step (1) is reclaimed is connected 2 with step) carrier framework that reclaims connects (Xho I and Sal I are isocaudarners), obtains recombinant plasmid pCAMBIA1300-ZmPHR1 ∷ GFP, and its structural representation is seen Fig. 2.

Implement the acquisition of 3 transgenic lines

1, the acquisition of restructuring agrobacterium strains

Utilize freeze-thaw method to import agrobacterium strains GV3101 recombinant plasmid pCAMBIA1300-ZmPHR1 ∷ GFP, obtain to contain the restructuring Agrobacterium of ZmPHR1 gene.

2, the acquisition of transfer-gen plant

(1) transforms the environmental Arabidopis thaliana of Colombia with restructuring Agrobacterium bacterium liquid by topical application.Obtain T0 for seed;

(2) screened for 2 generations with Totomycin (50mg/L);

(3) positive T2 is germinateed at the MS flat board that contains microbiotic (50mg/L Totomycin) for plant, 2-3 observes the resistance response situation after week, does not have positive strain of separating with feminine gender for turning the ZmPHR1 homozygous lines, i.e. T3 generation (seeing Fig. 3).

3, The evaluation of transgenosis homozygous lines

T3 detects through PCR and RT-PCR for plant, obtains 8 positive strains (Fig. 4, Fig. 5) of expressing ZmPHR1 at dna level and rna level.

Utilize the living imaging system, in the transgenosis homozygous lines, observe GFP fluorescence (Fig. 6), utilize the confocal fluorescent microscope in transgenosis is isozygotied the apical meristem cell's nuclear that is, to observe GFP fluorescence (Fig. 7).

Implement the acquisition of 4 overexpression transgenic lines

1, high phosphorus is processed

The T3 that upgrowth situation is identical places 23 ℃ of illumination boxs to carry out the high phosphorus nutrient solution for 30 strains of strain seedling and wild-type seedling and cultivates (3 days change one time of nutrition liquid) respectively.Nutritive medium is: 2 mM Ca (NO ₃) ₂, 0.65 mM MgSO ₄, 25 μ M Fe-EDTA, 5 μ M MnSO ₄, 50 μ M KCl, 2 μ M ZnSO ₄, 0.5 μ M CuSO ₄, 0.005 μ M (NH ₄) ₆Mo ₂₄With 25 m Μ H ₃BO ₄1mM KH2PO4。

2, the detection of ZmPHR1 gene expression amount in transgenic line

Extract the RNA of the Arabidopis thaliana overground part of high phosphorus processing, reverse transcription is cDNA; Carry out Real-time PCR amplification take cDNA as template.

The primer of amplification ZmPHR1 gene is as follows:

Forward primer: 5'-CACCCTTTATTTCTCAGTCATCCAA-3'

Reverse primer: 5'-TCATTTTGTGTAGCACTCTCATCAG-3'

Then can find out the relative expression quantity of overexpression strain and wild-type strain relatively to carry out after the same treatment in the overexpression plant expression amount of ZmPHR1 gene as 1, see Fig. 8.The expression amount of ZmPHR1 gene is higher than the wild-type plant under the high phosphorus treatment condition.The expression amount of the ZmPHR1 of T1 strain is high 13.06 times than the wild-type strain, thereby has obtained the transgenosis homozygous lines (Fig. 8) of overexpression ZmPHR1.

Implement the checking of 5 ZmPHR1 gene functions

1, different phosphate processing horizontal

Basic culture solution is composed as follows: 2 mM Ca (NO ₃) ₂, 0.65 mM MgSO ₄, 25 μ M Fe-EDTA, 5 μ M MnSO ₄, 50 μ M KCl, 2 μ M ZnSO ₄, 0.5 μ M CuSO ₄, 0.005 μ M (NH ₄) ₆Mo ₂₄With 25 m Μ H ₃BO ₄In the high phosphorus nutrient solution, KH2PO4 concentration is 1mM.Low-phosphorous nutrient solution is composed as follows: be consistent in order to make the K ionic concn in the nutritive medium, replenish 0.99 mM KCl, other same complete culture solution again; In the low-phosphorous nutrient solution, KH2PO4 concentration is 0.01mmol/L.

The T3 that upgrowth situation is identical places 23 ℃ of illumination boxs to carry out water planting (high phosphorus nutritive medium for 60 strains of strain seedling and wild-type seedling (WT) 60 strains respectively, changed one time of nutrition liquid in 3 days), cultivate 4-5 after week, 30 strains are used low-phosphorous nutrient solution instead and were cultivated 7 days, 30 strains continue to cultivate (changing once in 3 days) with the high phosphorus nutrient solution, then observe content of tatal phosphorus and the available phosphorus content of taking pictures and measuring plant amount of dry matter, plant shoot (leaf and stem) and root.

2, the form of overexpression strain and upgrowth situation

Compare high phosphorus and process, the growing way of overexpression strain and wild-type strain all is subject to inhibition (Fig. 9) in various degree under low-phosphorous condition.Plant bizet size and the amount of dry matter of overexpression strain all are higher than wild-type (table 1), and more obvious under low-phosphorous condition.Under low-phosphorous condition, the root/shoot ratio of overexpression strain is starkly lower than wild-type.Illustrate that the overexpression strain reduces the susceptibility of low-phosphorus stress, resistibility strengthens, and namely the overexpression plant has that growing way is better than wild-type (Fig. 9) under the low-phosphorous condition.

Table 1Overexpression strain and wild-type strain plant dry weight, root/shoot ratio and the content of tatal phosphorus under the different P levels condition

3, full phosphorus and available phosphorus content analysis

3.1 available phosphorus is measured, and utilizes molybdenum antimony resistance colorimetric method to carry out.

(1) reagent preparation

1) glacial acetic acid aqueous solution (volume ratio) (extraction agent) of preparation 100ml 2%.

2) preparation sulfuric acid-ammonium molybdate solution (solution A): the ammonium molybdate that namely contains 0.4% (W/V) among the 0.5N H2SO4

1. ((NH4) 6MO7O244H2O is in 100ml distilled water to dissolve the 0.8g ammonium molybdate;

2. the dense H2SO4 (98%) that then slowly adds 5ml;

3. abundant mixing adds water to 200ml after the cooling

3) ascorbic acid solution (solution B) of preparation 10% (W/V)

Dissolving 2g xitix adds water to 20ml at last in water, store in the brown bottle, in 4 ℃ of Refrigerator stores (preferably now with the current).

4) 100ml 0.5% antimony tartrate potassium solution

Add 0.5% antimony tartrate potassium solution 10ml in the 100mlA liquid.

5) working solution: by volume 6:1 mixed liquor A and solution B. annotate: working solution needs preparation every day, prepares rear placement and re-uses after 2 hours.

(2) operation steps

1) takes by weighing an amount of (about 0.1g) overground part or root and pack in the 2.0mL centrifuge tube, spare with the liquid nitrogen mill ,In 4 ℃ of placements (on ice or refrigerator) to the sample freeze thawing;

2) add 2mL 2% (volumn concentration) glacial acetic acid aqueous solution, temperature is bathed 30min in 42 ℃ of water-baths.

3) the centrifugal 2min of 12000rpm, supernatant liquor is used for the mensuration of available phosphorus content.

4) get the 1m1 working solution and mix with 1 ml sample supernatant liquor, in 37 ℃ of incubations 1 hour.

5) reaction solution is in cooled on ice or 4 ℃ of 5 min termination reaction.

Measure absorption value under the 820nm visible wavelength.

(3) making of phosphorus typical curve

Take by weighing 0.44g KH2PO4 (80 ℃ of dry 2h) and be dissolved in the distilled water, be settled to get mother liquor (inorganic phosphorus concentration is 100 μ g/mL)

Then be respectively the reference liquid of 0.2,0.4,0.6,0.8,1 and 2 μ g/mL with mother liquor configuration inorganic phosphorus concentration, distilled water is blank.

Get the above-mentioned reference liquids of 500 μ L (or distilled water), add 500 μ L AMES reaction solutions, 37 ℃ were reacted 1 hour; Place the 5min termination reaction for 4 ℃.Measure absorption value under the 820nm visible wavelength.And drawing standard curve.

(4) calculate

Plant available phosphorus (Pi) content (mg Pi/g FW)=OD value * (V/m) * (V2/V 1) * C

The mass concentration (PPM:mg/L) of phosphorus in the liquid to be measured after OD value one converts

V one sample prepares the ml number of solution, namely sample with the volume (this is 0.01 L) of extraction agent

M one sample fresh weight (g) (according to reality claim Mass Calculation)

V1 one draws reaction used volume (1 ml)

The overall product of V2 one reaction solution (3m1)

The extension rate of C one sample (if sample concentration is too high, need be diluted to again reaction behind 1 ml)

3.2 full phosphorus is measured

First material is carried out H at 300 ° of C ₂SO ₄-H ₂O ₂Disappear and boil, the recycling molybdenum antimony resistance colorimetric method is measured.

3.3 result of study

No matter under high phosphorus or low-phosphorous culture condition, the content of tatal phosphorus of overexpression strain is higher than the wild-type strain, but difference not obvious (table 1).And under low-phosphorous condition, the available phosphorus content in the overexpression strain cauline leaf then is the twice of wild-type, obvious difference.Available phosphorus content in root is then without significant difference (Figure 10).This result's hint, the overexpression strain can improve the available phosphorus content in the cauline leaf under low-phosphorous condition.

4, to the regulation and control of downstream phosphate starvation responsive genes and phosphate transporter gene

To the phosphate starvation responsive genes in overexpression strain and the wild-type strain AtIPS1, AtPS2, AtPS3With AtRNS1Expression carry out quantitative PCR detection.The result shows, under the low-phosphorous condition, compares the wild-type strain, in the ZmPHR1 overexpression strain root AtIPS1, AtPS2, AtPS3With AtRNS1Expression amount all got obvious raising (Figure 11 a, c, e, g); In cauline leaf, the expression amount of four kinds of genes also obtains raising, but only has AtPS2 and AtRNS1Rise apparent in view (Figure 11 c, g).Under the high phosphorus condition, in the ZmPHR1 overexpression strain root AtPS2, AtPS3With AtRNS1Expression amount also obtain raising (Figure 11 d, f, h).And no matter under the sort of phosphorus culture condition, AtRNS1Expression in the overexpression strain all is higher than wild-type strain (Figure 11 g, h).Above presentation of results ZmPHR1 has regulated and control AtIPS1, AtPS2, AtPS3With AtRNS1The expression of four kinds of genes.

To the phosphate transporter gene Pht1 in overexpression strain and the wild-type strain; 2, Pht1; 4, Pht1; 8 and Pht1; Quantitative PCR detection is carried out in 9 expression.Under low-phosphorous condition, the Pht1 in the overexpression strain root; 2, Pht1; 8 Pht1; 9 expression amount obtains raising (Figure 12 a, e, g), and under the high phosphorus condition, the Pht1 in the overexpression strain root; 2 and Pht1; 9 expression amount also is improved (Figure 12 b, h), in cauline leaf, and Pht1; 2, Pht1; 8 Pht1; 9 expression amount does not detect (Figure 12 a, b, e, f, g, h).Under low-phosphorous condition, Pht1; 2, Pht1; 8 and Pht1; 9 expression in overexpression strain root are higher than high phosphorus and process.Above result shows, under low-phosphorous condition, ZmPHR1Regulated and control Pht1 in the root; 2, Pht1; 8 and Pht1; 9 expression. ZmPHR1 mayTo Pht1; 4 expression does not have regulating and controlling effect (Figure 12 c, d).

SEQ ID NO：1

1 ATGAGGAAGT TTAATCTGAT GCAGTCTCAA AAGAGCAGAG TTTTGGGAGC AATGTCATCC

61 TCTTTGCCTA TTCTGCCAAA TCCTTTGAAA GGGAGCTTCC CAAGGCCTCA TAACCCCCAG

121 CATATTCCTA TGTTGAGGCA GCTGCCTGAT GACTCTATGC CCTTGTGTAT TGACACACAT

181 CAGTCTGTTA GTTTGCACCC AAGAGCTGGT GTTATCGGGG TACCATATTC AGGCTACACT

241 GCTAGTCCAC TTGATTCTGT GTCTAACCTT GATAGCCAGA CAATGGCTGC ACCCTTTATT

301 TCTCAGTCAT CCAATTTTGA AGCTCTCCAG TCTCTATCTG ATAATACCCC AGAAACACAC

361 ACTAAGGCAG CCTGGTTCAC ATCTTCTATG GATGTTTCAC CACTCAACAC AGATAATATT

421 GCTGCTTCTG ATGTTAATCA AATCCATAGT ATACGTCCTG CTATGACATC TGATGAGAGT

481 GCTACACAAA ATGATTGGTG GGCAGATATA ATGAATGATG ATTGGAAAGA TATTCTTGAC

541 GCAACAGCTA CTGATTCCCA CTCAAAAGCC ATGATTCAAA TTTCCAACTC AGCTACATCA

601 CTACCTGCAG TAAATCAGTC AGCTTCATCT CATAGTAGGG AGATTTGTCC TGTTGCTAGT

661 CCTCCCAATA GCAGCAATGC TTCAGTTGCC AAACAACGGA TGAGATGGAC CCCAGAACTC

721 CATGAATGTT TTGTAGATGC TGTAAATCAG CTTGGCGGTA GCGAAAAAGC TACTCCTAAG

781 GGTGTGCTAA AGCTTATGAA AGTTGATGGT TTGACTATAT ATCATGTCAA AAGCCACCTG

841 CAGAAGTACC GCACAGCCCG CTATAAACCA GACCTGTCAG AAGGTACATC GGAAAAAAGG

901 ACAGCCACTG AAGAGCTGGT CCTGGACCTG AAAACGAGCA TGGATCTTAC TGAAGCGCTG

961 CGCCTTCAGA TGGAAGTCCA GAAACGGCTT CATGAACAGC TTGAGATTCA GAGAAAATTG

1021CAGTTGCGGA TTGAAGAGCA AGGAAAGTAT CTGCAGATGA TGTTTGAAAA GCAGAGTCAA

1081TCGAGCACGG AGAAAGTCCA GGATCCATCC TCAAGGGATA CAACAGCTAA ACCGTCATCT

1141AATCAGAGCC AGTCTACAAA CAAGGATAGT GGTGCAACCA TGGACCCAAA TGGAACGGGA

1201GACATCGCAC GGACTGCAGA ACTGGGAGAA CGTTCGTCTG AACTAGGTGT AAAACAGAAA

1261CTTGTAGAGA TCGAATCTGG TACCGAAGTA GCCACAGGCG ACAGATCTAA GATCTCCCAA

1321GAGAAGCGGC GCAAACTGCA AGATAGTTAA

SEQ ID NO：2

1 MRKFNLMQSQ KSRVLGAMSS SLPILPNPLK GSFPRPHNPQ HIPMLRQLPD DSMPLCIDTH

61 QSVSLHPRAG VIGVPYSGYT ASPLDSVSNL DSQTMAAPFI SQSSNFEALQ SLSDNTPETH

121 TKAAWFTSSM DVSPLNTDNI AASDVNQIHS IRPAMTSDES ATQNDWWADI MNDDWKDILD

181 ATATDSHSKA MIQISNSATS LPAVNQSASS HSREICPVAS PPNSSNASVA KQRMRWTPEL

241 HECFVDAVNQ LGGSEKATPK GVLKLMKVDG LTIYHVKSHL QKYRTARYKP DLSEGTSEKR

301 TATEELVLDL KTSMDLTEAL RLQMEVQKRL HEQLEIQRKL QLRIEEQGKY LQMMFEKQSQ

361 SSTEKVQDPS SRDTTAKPSS NQSQSTNKDS GATMDPNGTG DIARTAELGE RSSELGVKQK

421 LVEIESGTEV ATGDRSKISQ EKRRKLQDS

Claims (10)

1. a low phosphorus response regulatory factor ZmPHR1 is characterized in that, its aminoacid sequence is shown in SEQ ID NO:2, or this sequence is through replacing, lack or adding the formed aminoacid sequence with same function of one or several amino acid.

One kind the coding low-phosphorus stress factor Z mPHR1 claimed in claim 1 gene.

3. gene claimed in claim 2 is characterized in that, the sequence of described gene contains the nucleotide sequence shown in the SEQ ID NO:1.

4. the primer of the total length of increase claim 2 or 3 described genes or its any fragment pair, described primer sequence is as follows:

Primer is to 1

Forward primer: 5 '-AAGCAAGGTAATGAAATG-3 '

Reverse primer: 5 '-TGAATAGTGCAACCGATA-3 ';

Primer is to 2

Forward primer: 5 '-CACCCTTTATTTCTCAGTCATCCAA-3 '

Reverse primer: 5 '-TCATTTTGTGTAGCACTCTCATCAG-3 '.

5. the plant expression vector that contains claim 2 or 3 described genes.

6. plant expression vector claimed in claim 5, it is characterized in that, described carrier is pJIT163-ZmPHR1 ∷ GFP, and the various intermediate carriers that generate in building process, is described gene to be inserted between the SalI of pJIT163 ∷ GFP and the BamH I restriction enzyme site obtain.

7. plant expression vector claimed in claim 5, it is characterized in that, described carrier is pCAMBIA1300-ZmPHR1 ∷ GFP, and the various intermediate carriers that in building process, generate, be that the small segment that restriction enzyme KpnI is connected with XhoI double digestion recombinant plasmid first is obtained with the large fragment connection that restriction enzyme KpnI is connected with SalI double digestion pCAMBIA1300.

8. an agrobacterium strains that contains the described plant expression vector of claim 7 is characterized in that, contains plant expression vector pCAMBIA1300-ZmPHR1 ∷ GFP, recombinant plasmid second is imported agrobacterium strains GV3101 by freeze-thaw method obtain.

9. containing the transgenosis homozygous lines of the overexpression of the described plant expression vector of claim 7, it is characterized in that, is to import in the Arabidopis thaliana through topical application by the Agrobacterium bacterium liquid that will contain plant expression vector pCAMBIA1300-ZmPHR1 ∷ GFP to obtain.

10. claim 2 or 3 application of described gene in plant breeding.

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