CN103360482A - Transcriptional active protein AtPHD6 as well coding gene and application thereof - Google Patents

Abstract

The invention discloses a transcriptional active protein AtPHD6 as well as a coding gene and application thereof. The protein provided by the invention is a protein with one of the following amino acid residue sequences: (a) a protein consisting of an amino acid sequence shown by a sequence 2 in a sequence table, and (b) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues on the amino acid sequence shown by the sequence 2 in the sequence table, is related to plant stress tolerance and is derived from the sequence 2. The transcriptional active protein AtPHD6 has an important value for culturing stress tolerance plant varieties in particular new varieties including abiotic stress tolerance (salt tolerance) crops, forest grasses and the like, can be used for culturing and identifying the stress tolerance plant varieties needed by agriculture and animal husbandry and ecological environmental control, and is of great significance for improving the crop yield.

Description

Transcriptional activity albumin A tPHD6 and encoding gene thereof and application

Technical field

The present invention relates to biological technical field, relate in particular to a kind of transcriptional activity albumin A tPHD6 and encoding gene and application.

Background technology

The variation of physics, chemical factor in the environment, such as Stress Factors such as arid, saline and alkaline, low temperature growing of plant had material impact, can cause the extensive underproduction of farm crop when serious, therefore cultivating the high crop of resistance of reverse is one of major objective of plant husbandry.At present, using gene engineering technique carries out breeding and has become one of important method that improves the crop resistance of reverse.Higher plant cell has many approach and replys various environment stresses in the environment, and its transcription factor plays a part the regulation and control effector of anti-the retrocorrelation expresses.Now in plant, found the multiclass transcription factor relevant with plant stress tolerance, for example: the DREB class among the EREBP/AP2, bZIP, MYB etc.The albumen that PHD (Plant Homodomain) class has a transcriptional activity extensively is present in animals and plants and bacterium, the viral protein, such as mammiferous CBP proteinoid, and people's ING1 family, yeast Yng2p albumen, viral MIR1 albumen.PHD proteinoid structural domain is the zinc fingers of C4HC3 class, and its function is mainly following a few class: (1) participates in the relevant transcriptional control of chromatin as acetylize or deacetylase; (2) participate in proteolytic degradation as the E3 ubiquitin ligase; (3) participate in the relevant transcriptional control of chromatin as PIPs acceptor perception PIPs signal.Found first the PHD proteinoid in Arabidopis thaliana, the functional study of this proteinoid is still few in the plant.

Soybean is one of China five large crops, understands fully its abiotic stress tolerance molecule mechanism, and then provides the basis for improving its resistance of reverse, has important theory and realistic meaning.

Summary of the invention

An object of the present invention is to provide a kind of transcriptional activity albumin A tPHD6 and encoding gene thereof.

Albumen provided by the present invention, name is called AtPHD6, derives from (Arabidopsis thaliana cv Columbia-0, Col-0), is the protein with one of following amino acid residue sequences:

(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;

(b) with the aminoacid sequence shown in the sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with the plant stress tolerance protein that is derived by sequence 2.

Wherein, the sequence 2 in the sequence table is comprised of 260 amino-acid residues, belongs to the PHD family in the Arabidopis thaliana.The reading frame of its encoding gene comprises 783 Nucleotide.

In the above-mentioned albumen, the replacement of described one or several amino-acid residue and/or disappearance and/or interpolation refer to replacement and/or disappearance and/or the interpolation of no more than ten amino-acid residues.

The encoding gene AtPHD6 of above-mentioned albumin A tPHD6 also belongs to protection scope of the present invention.

The encoding gene of above-mentioned albumin A tPHD6 is the dna molecular of following (1) or (2) or (3):

(1) dna molecular shown in the sequence 1 in the sequence table;

(2) the dna sequence dna hybridization that under stringent condition, limits with (1) and the dna molecular of coded plant stress tolerance correlative protein;

(3) dna sequence dna that limits with (1) has 70% at least, have at least 75%, have at least 80%, have at least 85%, have at least 90%, have at least 95%, have at least 96%, have at least 97%, have at least 98% or have at least a dna molecular of 99% homology and coded plant stress tolerance correlative protein.

In the above-mentioned encoding gene, described stringent condition can be as follows: 50 ℃, and at 7% sodium lauryl sulphate (SDS), 0.5M NaPO ₄With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 2 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M NaPO ₄With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 1 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M NaPO ₄With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 0.5 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M NaPO ₄With hybridize in the mixing solutions of 1mM EDTA, at 50 ℃, 0.1 * SSC, rinsing among the 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M NaPO ₄With hybridize in the mixing solutions of 1mM EDTA, at 65 ℃, 0.1 * SSC, rinsing among the 0.1%SDS; Also can be: at 6 * SSC, in the solution of 0.5%SDS, 65 ℃ of lower hybridization, then use 2 * SSC, 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.

Wherein, the sequence 1 in the sequence table is comprised of 783 deoxynucleotides, and this sequence is the reading frame of AtPHD6 gene, and coding has the protein of the amino acid residue sequence of sequence 2 in the sequence table.

The recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contain above-mentioned encoding gene all belong to protection scope of the present invention.

Above-mentioned recombinant vectors is specially the recombinant vectors that obtains between the BamH I of above-mentioned encoding gene insertion pROKII carrier and Kpn I recognition site in an embodiment of the present invention.

The primer pair of described full length gene or its any fragment of increasing also belongs to protection scope of the present invention.

Above-mentioned primer pair specifically can be following 1 in an embodiment of the present invention) or 2):

1) primer pair that is formed by DNA shown in the sequence 4 of DNA shown in the sequence 3 of sequence table and sequence table;

2) primer pair that is formed by DNA shown in the sequence 6 of DNA shown in the sequence 5 of sequence table and sequence table.

Above-mentioned albumen or its encoding gene or the application in the regulating plant resistance of reverse of above-mentioned recombinant vectors, expression cassette, transgenic cell line or recombinant bacterium are the scope of protection of the invention.

In the above-mentioned application, described regulating plant resistance of reverse is specially the raising plant stress tolerance;

Described anti-against further being specially drought-enduring and/or salt tolerant;

Described plant is specially dicotyledons or monocotyledons, and described dicotyledons further is specially Arabidopis thaliana.

Second purpose of the present invention provides a kind of cultivation transgenic plant method.

Method provided by the invention is for the encoding gene with described albumen imports the purpose plant, obtains transgenic plant, and the resistance of reverse of described transgenic plant is higher than described purpose plant.

In aforesaid method, described anti-contrary be drought-enduring and/or salt tolerant.

In aforesaid method, the encoding gene of described albumen imports the purpose plant by above-mentioned recombinant vectors;

In aforesaid method, described salt tolerance is by improving survival rate, increase the plant fresh weight and/or reducing relative ion permeability embodiment;

Above-mentioned plant fresh weight is that plant shoot divides fresh weight.

In aforesaid method, described drought tolerance embodies by improving plant height;

In aforesaid method, described plant is dicotyledons or monocotyledons, and described one step of dicotyledons is specially Arabidopis thaliana.

Above-mentioned transgenic plant are interpreted as and not only comprise the first-generation transgenic plant that described gene transformation purpose plant is obtained, also comprise its filial generation.For transgenic plant, can in these species, breed this gene, also available traditional breeding method enters this transgenosis other kind of same species, in commercial variety.Described gene being imported the purpose plant, can make in the described protein purpose plant syntheticly, and then is that the resistance of reverse proterties of purpose plant is improved.

Said gene can be carried out first following modification, imports again among the host, to reach better expression effect:

1) modifies according to actual needs and optimize, so that gene efficient expression; For example, the codon that can have a preference for according to recipient plant is keeping nucleotide sequence coded amino acid whose its codon that changes simultaneously of the present invention to meet plant-preference; In the optimizing process, preferably can make to keep certain GC content in the encoding sequence after the optimization, to realize best the high level expression of quiding gene in the plant, wherein GC content can be 35%, be preferably more than 45%, more preferably more than 50%, most preferably more than approximately 60%;

2) modify the gene order of contiguous initial methionine, so that translate effectively initial; For example, utilization known effective sequence in plant is modified;

3) be connected with the promotor of various expression of plants, be beneficial to its expression in plant; Described promotor can comprise that adjusting, Chemical Regulation are regulated, grown to composing type, induction type, sequential, tissue is preferred and tissue-specific promoter; The selection of promotor will be along with expression time and space requirement and is changed, and depends on the target species; For example tissue or the specific expressing promoter of organ, acceptor in what period of growing is decided as required; Although having proved the many promotors that derive from dicotyledons is operational in monocotyledons, vice versa, but ideally, select the dicotyledons promotor to be used for the expression of dicotyledons, monocotyledonous promotor is used for the expression of monocotyledons;

4) with the Transcription Termination sub-connection that is fit to, also can improve the expression efficiency of gene of the present invention; For example derive from the tml of CaMV, derive from the E9 of rbcS; Any known available terminator that works in plant can be connected with gene of the present invention.

5) introduce enhancer sequence, such as intron sequences (for example deriving from Adhl and bronzel) and virus leader sequence (for example deriving from TMV, MCMV and AMV).

In actually operating, also gene of the present invention can be carried out the cell-targeting location.Can utilize the existing technology in this area to realize.For example, target-gene sequence and the gene order of the present invention that derives from the targeted cells device merged, import again in the vegetable cell, just can locate.

Carry described gene expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity be led, conventional biological method transformed plant cells or the tissue such as agriculture bacillus mediated by using, and the plant tissue that transforms is cultivated into plant.

The expression of the AtPHD6 of experiment showed, of the present invention is subjected to high salt, polyoxyethylene glycol (6000) simulating drought and low temperature induction, also is subjected to inducing of Abscisic Acid ABA.It is relevant that known ABA and plant abiotic stress are replied, so AtPHD6 may be relevant with the regulation and control that plant is replied abiotic stress.Afterwards with the mediation of AtPHD6 gene through Agrobacterium, import among the Arabidopis thaliana Col0, obtained the pure lines strain of overexpression AtPHD6 gene, above-mentioned transgenic line is compared with the wild-type Arabidopis thaliana, its salt tolerance is significantly increased, and illustrates that the AtPHD6 gene can significantly improve the salt tolerance of plant.The regulation and control that the AtPHD6 involved in plant is replied abiotic stress.

The present invention is for cultivating the plant with adverse resistance kind, the new variety such as crop, woods grass of particularly cultivating abiotic stress tolerance (salt tolerant) have important value, can be used for cultivation and the evaluation of the required resistance of reverse plant variety of husbandry and ecological environment treatment, significant to improving crop yield.And proved the regulation and control that the certain involved in plant of some members the PDH family is replied abiotic stress from the molecular biology angle, it is expressed and the abiotic stress tolerance of plant is proportionate.

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

Description of drawings

Fig. 1 is that the AtPHD gene is transcribed feature when different treatment

Fig. 2 is the schematic diagram of plant expression vector pROK II-AtPHD6

Fig. 3 is the evaluation of AtPHD6 mutant phd6

Fig. 4 was the relative expression quantity of AtPHD6 in expression, mutant and the adjoining tree

Fig. 5 is that AtPHD6 crosses the salt tolerance comparison of expressing strain, mutant and contrast

Fig. 6 is the survival rate comparison after AtPHD6 crosses expression strain and mutant salt stress

Fig. 7 is that the over-ground part fresh weight compared after AtPHD6 crossed expression strain and mutant salt stress

Fig. 8 is transfer-gen plant and the relative ion permeability of contrast behind salt stress

Fig. 9 is that the drought tolerance of transfer-gen plant, mutant and contrast is identified

Embodiment

Employed experimental technique is ordinary method if no special instructions among the following embodiment.

Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.

% among the following embodiment if no special instructions, is the quality percentage composition.Quantitative test in following examples all arranges repeated experiments three times, and data are mean value or the mean+SD of three repeated experiments.

The illumination that all vegetable materials all grow in 22 ℃ of every days is 16h/8h (illumination/dark).

PROKII carrier (binary expression vector) is documented in D.C.Baulcombe, G.R.Saunders, M.W.Bevan, M.A.Mayo and B.D.Harrison, Expression of biologically active viral satellite RNA from the nuclear genome of transformed plants.Nature 321 (1986), among the pp.446-449, the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity.

Agrobacterium GV3101 bacterial strain is documented in Clough-SJ, Bent-AF.Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Plant-Journal.1998,16:6, among the 735-743, the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity.

The environmental Arabidopis thaliana (col-0) of Colombia: seed is available from Arabidopsis Biological Resource Center (ABRC), hereinafter to be referred as the wild-type Arabidopis thaliana.

The screening of embodiment 1, soybean AtPDH6 and encoding gene thereof and the clone of cDNA thereof

1, the acquisition of soybean AtPDH6 and encoding gene thereof

Early stage studies show that PHD protein family involved in plant with transcriptional activity to the responsing reaction of abiotic stress, and relevant with plant stress tolerance.By Blast search arabidopsis gene group database, cluster the PHD genoid, its coding comprises the zinc finger protein of PHD structural domain, has 7, called after AtPHD1-7.Wherein AtPHD6 has the nucleotide sequence of sequence 1 in the sequence table, the amino acid residue sequence that coding has sequence 2 in the sequence table.Amino acid sequence analysis shows, sequence 2 is comprised of 260 amino-acid residues in sequence table.

Design primer according to the AtPHD6 gene order:

AtPHD6 sense:5 '-ATGGAAGGAGGAACTGCGCAC-3 ', (sequence 3)

AtPHD6 antisense:5 '-CTAGGGTCGTGCTCTTTTGTT-3 ' (sequence 4)

Use the RT-PCR method, amplification AtPHD6 gene from the total RNA of Arabidopis thaliana.Get the wild-type Arabidopsis leaf, place liquid nitrogen to grind, be suspended from the 4mol/L sulphur hydracid guanidine, and with acid phenol, chloroform extracting, adding dehydrated alcohol in the supernatant precipitates, and will precipitate at last total RNA that obtains soluble in water.Get the total RNA of 5 μ g and carry out reverse transcription with reverse transcription test kit (Promega company) by the method for test kit, carry out pcr amplification reaction take the cDNA fragment that obtains as template.20 μ l PCR reaction systems are: 1 μ l, one chain cDNA (0.05 μ g), 1 μ l primer (20 μ M), 2 μ l, 10 * PCR damping fluid, 0.4 μ l dNTP (10mM) and 1U Taq archaeal dna polymerase, supply 20 μ l with ultrapure water, liquid level Covering Liguid paraffin oil.Reaction is carried out at PE9600 type PCR instrument, and its program is 94 ℃ of sex change 5min; 94 ℃ of 1min again, 56 ℃ of 1min, 72 ℃ of 1min, altogether 30-32 circulation; Then 72 ℃ are extended 10min; 4 ℃ of preservations.The sequential analysis after reclaiming of 783bpPCR product, the result shows the nucleotide sequence that this PCR product has sequence 1 in the sequence table, then is cloned in the multiple clone site of pMD18-T plasmid, obtains recombinant vectors pMDAtPHD6.

2, the expression characteristic of AtPHD6 gene under abiotic stress

Wild-type Arabidopis thaliana Col0 is carried out arid, NaCl, ABA and the subzero treatment of polyoxyethylene glycol (PEG) simulation, be used for analyzing Arabidopis thaliana AtPHD6 and under abiotic stress and dormin (ABA) are processed, transcribe feature.The Arabidopis thaliana seed in basin, after 2 weeks of growing, is carried out respectively following Stress treatment to seedling:

Arid is processed: the root system of Arabidopsis thaliana Seedlings is placed 20%PEG solution, and arid is cultivated sampling after 0 hour, 1 hour, 3 hours, 6 hours and 12 hours under illumination condition respectively.

Salt is processed: the root system of seedling is placed 200mM NaCl solution, take a sample after 0 hour, 1 hour, 3 hours, 6 hours and 12 hours in illumination cultivation respectively.

ABA processes: the root system of seedling is placed 100 μ M ABA, take a sample after 0 hour, 1 hour, 3 hours, 6 hours and 12 hours in illumination cultivation respectively.

Subzero treatment: seedling is placed 0 ℃ of incubator, take a sample after 0 hour, 1 hour, 3 hours, 6 hours and 12 hours in illumination cultivation respectively.

The extraction above-mentioned 1 of total RNA.Use RT-PCR and analyze the AtPHD6 gene transcribe feature when above-mentioned processing, the primer is with above-mentioned 1.The result as shown in Figure 1, transcribing of AtPHD6 gene is subjected to inducing of high salt, arid and low temperature stress, what be subjected to also simultaneously that dormin ABA processes induces.The above results shows that AtPHD6 possibility involved in plant is to the responsing reaction of abiotic stress.

The application of embodiment 2, AtPHD6

One, the acquisition of overexpression AtPHD6 gene Arabidopis thaliana strain

1, the structure of recombinant expression vector pROKII-AtPHD6

The cDNA (sequence 1) of the AtPHD6 gene that obtains take embodiment 1 is as template, with containing restriction enzyme BamHI and Kpn I double digestion carries out pcr amplification by Primer-F+ and Primer-R-as primer, acquisition contains the PCR product of goal gene fragment, after the order-checking evaluation is errorless, with above-mentioned PCR product through BamHI and KpnI double digestion, this enzyme that obtains is cut product and is connected with the carrier framework that the expression vector pROKII that cuts plant through same enzyme obtains, and obtains connecting product.To connect product and change in the intestinal bacteria, obtain transformant.Extract the plasmid of transformant, send to order-checking, this plasmid is the carrier that obtains between the BamHI of the sequence 1 insertion pROKII in the sequence table and the KpnI restriction enzyme site, and with this carrier called after pROKII-AtPHD6, and the sequence in the sequence table 1 is positioned at after the CaMV 35S promoter.Recombinant expression vector pROKII-AtPHD6 structural representation as shown in Figure 2.

Primer-F+:5 '-GAGGGATCCATGGAAGGAGGAACTGCGCAC-3 ' (sequence 5)

Primer-R-:5 '-GAGGGTACCCTAGGGTCGTGCTCTTTTGTT-3 ' (sequence 6)

2, the evaluation of AtPHD6 mutant phd6

(Arabidopsis Biological Resource Center, the ABRC numbering: salk_075676), Fig. 3 A has shown the insertion point of T-DNA among the mutant phd6 to T-DNA insertion mutation body phd6 available from ABRC.

With wild-type Arabidopis thaliana and phd6 mutant seed after 2 weeks of culture dish growth, transplant (illumination that grows in 22 ℃ of every days is 16h/8h (illumination/dark)) to the basin after blooming, get complete stool and extract RNA and be RT-PCR and identify.The same Primer-F+ of primer (sequence 5) and Primer-R-(sequence 6), the result can find out shown in Fig. 3 B, fails to detect transcribing of AtPHD6 gene in the phd6 mutant, verifies that further this mutant is by the AtPHD6 deletion mutant.

3, the evaluation of overexpression AtPHD6 gene Arabidopis thaliana strain

Recombinant vectors pROK II-AtPHD6 is imported among the Agrobacterium GV3101 with the electric shock conversion method, obtain recombinant bacterium, extract the plasmid of recombinant bacterium, order-checking, the result contains the recombinant bacterium called after GV3101/pROKII-AtPHD6 of this plasmid for this plasmid is pROKII-AtPHD6.

Single bacterium colony of picking GV3101/pROKII-AtPHD6 in 28 ℃ of cultivations 8 hours, is transferred to and continues among the 200mlLB to cultivate 3 hours in 5mlLB, is resuspended in the LB substratum behind the receipts bacterium and obtains conversion fluid.The flower of wild-type Arabidopis thaliana (Arabidopsis thaliana) Col-0 is soaked in the conversion fluid 10 seconds, puts into MS substratum lucifuge after the taking-up and cultivated 8 hours, obtain T ₀For transformed the seed, it is sowed on the MS substratum that contains kantlex (50mg/L), obtain 28 strain T ₀In generation, turn the AtPHD6 Arabidopis thaliana.Screen 2 transgenic lines for further study, called after A6-6 and A6-10.

Extract above-mentioned A6-6 and A6-10T ₀In generation, turn the RNA of AtPHD6 Arabidopis thaliana Plant Leaf, and reverse transcription acquisition cDNA, carries out Real Time PCR and identify, probe is:

AtPHD6-Real+：5’-ACTGCGCACTACAGCCCTCG

AtPHD6-Real-：5’-TGCCGAGTGCAGGTTCGGGA

Take wild-type Arabidopis thaliana Col-0 as contrast.

The result as shown in Figure 4, wherein, among AtPHD6 overexpression transgenic line A6-6 and the A6-10, the relative expression quantity of AtPHD6 is respectively 14.2 ± 0.7 and 7.5 ± 0.5; The relative expression quantity of AtPHD6 is the expression of the original AtPHD6 of Arabidopis thaliana in the wild-type contrast (Col0), is 1.0 ± 0.1; And do not detect the expression of AtPHD6 among the mutant phd6, illustrate that A6-6 and A6-10 are for through identifying positive T ₁In generation, turn the AtPHD6 Arabidopis thaliana.

With T ₁Individual plant results seed, this is T ₂For seed, each single-strain seed is sowed respectively, continues screening to observe T with kantlex ₂The separation case in generation so repeats screening, until T ₃In generation, obtain the transgenic line of inheritance stability, and what obtain altogether genetic stability turns AtPHD6 gene strain.Comprising in order to carry out the T of further experiment ₃In generation, turn AtPHD6 Arabidopis thaliana A6-6 and T ₃In generation, turn AtPHD6 Arabidopis thaliana A6-10 strain.

Adopting uses the same method imports empty carrier pROKII in the wild-type Arabidopis thaliana, obtains T ₀In generation, turn the empty carrier Arabidopis thaliana, carries out RT-PCR with above-mentioned AtPHD6-Real and AtPHD6-Real and identify, AtPHD6 does not express as a result, is illustrated as positive T ₀In generation, turn the empty carrier Arabidopis thaliana, and screening obtains T ₃In generation, turn the empty carrier Arabidopis thaliana.

Two, the salt tolerant of AtPHD6 gene overexpression strain and mutant, drought-enduring evaluation

Experiment sample is as T ₃In generation, turn empty carrier Arabidopis thaliana, T ₃In generation, turn AtPHD6 Arabidopis thaliana A6-6, T ₃In generation, turn AtPHD6 Arabidopis thaliana A6-10 strain and AtPHD6 mutant phd6 and wild-type Arabidopis thaliana Col0.

1, Salt-Tolerance Identification:

1) survival rate

Experiment sample following (15 seeds of each strain): T ₃In generation, turn empty carrier Arabidopis thaliana, T ₃In generation, turn AtPHD6 gene plant (two strains of A6-6 and A6-10), mutant phd6 and wild-type Arabidopis thaliana (Col0, contrast).

The seed of each strain plant is sowed simultaneously on the MS flat board, sprout after rear 5 days and seedling to be transplanted to respectively on the 1/2MS substratum that contains 200mM NaCl growth 10 days, the seedling replanting of NaCl processing after 10 days recovered growth 14 days under normal operation in vermiculite, relatively phenotype.Be treated to contrast (upper figure) with normal condition.

The result as shown in Figure 5, upper figure be do not carry out processing with normal condition growth, figure below recovered again 14 days after processing through NaCl, can find out, normal condition is processed, each strain growth is without significant difference.After 200mM NaCl processed, the growth of seedling of each strain all was subject to severe inhibition, and mutant is wilted serious, and contrast is taken second place, and AtPHD6 crosses the expression strain and substantially still survives.

Statistics renewal cultivation the 14th day survival rate, result can find out as shown in Figure 6,

The survival rate of A6-6, A6-10, phd6 and Col0 is about respectively 90%, 95%, 18%, 74%, shows, the plant of excessively expressing the AtPHD6 gene is apparently higher than wild-type, and mutant phd6 significantly is lower than wild-type.

2) over-ground part fresh weight

Above-mentioned 1) renewal cultivation detection in the 14th day over-ground part fresh weight, the result as shown in Figure 7, can find out, the over-ground part fresh weight of A6-6, A6-10, phd6 and Col0 is about respectively 0.24g, 0.21g, 0.1g and 0.04g, cross the fresh weight of the above-ground plant parts of expressing the AtPHD6 gene apparently higher than wild-type, and mutant phd6 significantly is lower than wild-type.

T ₃For turning empty carrier Arabidopis thaliana and wild-type Arabidopis thaliana result without significant difference.

3) relative ion permeability

Ion permeability is to detect the relative ionic weight that plant discharges when being subject to environment stress relatively, the degree that the damaged membrane at the expression position of identifying is hindered.In general, when if the damage of adverse circumstance cell membrane is large, Ion leakage in the cell is also large, the relative conductivity that then determines is larger, if and plant has certain resistance of reverse, the ion of preservation self that then can larger limit, thereby the ion that discharges is less, Ion leakage is few, and then relative conductivity is less.Therefore the Relative electro-conductivity rate of permeation has represented relative ion permeability, with the abiotic stress tolerance ability of plant, comprises salt tolerance, drought tolerance, low temperature resistant etc. presents certain dependency.

Experiment sample following (each strain 15 strain): T ₃In generation, turn empty carrier Arabidopis thaliana, T ₃In generation, turn AtPHD6 gene plant (two strains of A6-6 and A6-10), the environmental Arabidopis thaliana (Col-0) of mutant phd6 and Colombia.

Each strain plant aseptic seedlings in 3 age in week is immersed in the 200mM NaCl aqueous solution, measures respectively relative ion permeability after 48 hours.In the MS substratum, to soak in contrast (normal condition).

Relative ion permeability measuring method: the seedling of processing is cleaned 6 times with clear water, fully clean the ion of plant epidermis, plant is placed the 15ml Glass tubing, add the 12ml deionized water, 0.1Mpa vacuumize 30 minutes, room temperature is placed 3h, measures the specific conductivity G1 in solution this moment; Place boiling water to boil 30 minutes Glass tubing, treat the complete flavescence of blade, take out, temperature is measured specific conductivity G2 after dropping to room temperature again.Relative ion permeability=G2/G1.

The result as shown in Figure 8, in the MS substratum, the relative ion permeability that two AtPHD6 cross expression strain (A6-6 and A6-10) and wild-type Arabidopis thaliana is all very low, does not almost have difference.

After processing 2 days through 200mM NaCl, the relative ion permeability of wild-type Arabidopis thaliana and transgenic line all has rising in various degree, wild-type Arabidopis thaliana (Col0) relatively ion permeability is 66 ± 5%, and the relative ion permeability of transgenic line A6-6 is 43 ± 7%, and the relative ion permeability of A6-10 is 13 ± 1%.Illustrate that the damage of AtPHD6 overexpression strain cytolemma under high-salt stress is far below contrast.

T ₃For turning empty carrier Arabidopis thaliana and wild-type Arabidopis thaliana result without significant difference.

2, drought tolerance is identified

With T ₃In generation, turn empty carrier Arabidopis thaliana, mutant (phd6), wild-type (Col0) and T ₃The generation turn AtPHD6 gene plant (A6-6 and A6-10) seed in the 1/2MS substratum, vernalization 3 days.Grew 7 days for 23 ℃, sprigging is entered in the vermiculite, the week of growing stops to water, and after 14 days, the rehydration growth was added up plant height after 10 days.To coerce in contrast without drought.Each strain 15 strain.

The result as shown in Figure 9, A, the seedling that grew 14 days stops to water 14 days, 10 days phenotype of rehydration growth, wherein upper figure coerces without drought, figure below is afterwards rehydration 10 days of arid processing; B, the plant height statistics of rehydration after 10 days after arid is processed; As can be seen from Figure 9A, mutant under without non-irrigated stress state a little more than wild-type and two transgenic lines, but no significant difference (the upper figure of 9A); And mutant is obviously short in wild-type after the arid processing rehydration, and two transgenic lines all are significantly higher than wild-type (9A figure below).

Rehydration each strain plant height after 10 days after concrete arid is processed, the result can find out shown in Fig. 9 B,

The plant height of coercing without drought is about respectively: phd6 is 11.78cm; Col0 is 10.88cm; A6-6 is 11.4cm; A6-10 is 10.88cm;

Rehydration was after 10 days after arid was processed, and the plant height of different samples is about respectively: phd6 is 5.39cm; Col0 is 6.33cm; A6-6 is 7.42cm; A6-10 is 8.84cm.

T ₃For turning empty carrier Arabidopis thaliana and wild-type Arabidopis thaliana result without significant difference.

Comprehensive the above results, AtPHD6 has participated in the regulation and control of plant under high salt and drought stress.The overexpression of its encoding gene AtPHD6 has improved salt tolerant and the drought tolerance of transfer-gen plant.

Claims (10)

1. albumen is following (a) or (b):

(a) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 2;

(b) with the aminoacid sequence shown in the sequence in the sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with the plant stress tolerance protein that is derived by sequence 2.

2. the gene of coding claim 1 described albumen.

3. gene as claimed in claim 2, it is characterized in that: described gene is the dna molecular of following (1) or (2) or (3):

(1) dna molecular shown in the sequence 1 in the sequence table;

(2) the dna sequence dna hybridization that under stringent condition, limits with (1) and the dna molecular of coded plant stress tolerance correlative protein;

(3) dna sequence dna that limits with (1) has 70% at least, have at least 75%, have at least 80%, have at least 85%, have at least 90%, have at least 95%, have at least 96%, have at least 97%, have at least 98% or have at least a dna molecular of 99% homology and coded plant stress tolerance correlative protein.

4. the recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contain claim 2 or 3 described encoding genes.

5. recombinant vectors as claimed in claim 4 is characterized in that:

Described recombinant vectors is that the encoding gene of the described albumen of claim 1 is inserted in the pROKII carrier, obtains expressing the recombinant vectors of the described albumen of claim 1.

6. the primer pair of claim 2 or 3 described full length genes or its any fragment of increasing.

7. the described albumen of claim 1, claim 2 or 3 described encoding genes or the described recombinant vectors of claim 4, expression cassette, transgenic cell line or the application of recombinant bacterium in the regulating plant resistance of reverse;

Described regulating plant resistance of reverse is specially the raising plant stress tolerance;

Described anti-against further being specially drought-enduring and/or salt tolerant;

Described plant is specially dicotyledons or monocotyledons, and described dicotyledons further is specially Arabidopis thaliana.

8. a method of cultivating transgenic plant for the encoding gene with the described albumen of claim 1 imports the purpose plant, obtains transgenic plant, and the resistance of reverse of described transgenic plant is higher than described purpose plant.

9. method according to claim 8 is characterized in that: the described anti-contrary drought-enduring and/or salt tolerant that is.

10. it is characterized in that according to claim 8 or 9 described methods:

The encoding gene of the described albumen of claim 1 imports the purpose plant by claim 4 or 5 described recombinant vectorss;

Described salt tolerance is by improving survival rate, increase the plant fresh weight and/or reducing relative ion permeability embodiment;

Described drought tolerance embodies by improving plant height;

Described plant is dicotyledons or monocotyledons, and described one step of dicotyledons is specially Arabidopis thaliana.

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