CN1908011A - Plant inverse-resistant zinc finger protein, coding gene and application thereof - Google Patents

Abstract

The invention discloses a plant anti-inverse zinc protein and coded gene and application, which is characterized by the following: (a) protein of residue sequence of amino acid in the sequence list 2; (b) protein of (a) derivatized plant in the sequence list 2; improving resisting property for plant.

Description

A kind of plant stress-resistance zinc finger protein and encoding gene and application

Technical field

The present invention relates to a kind of plant stress-resistance zinc finger protein and encoding gene and application in the plant biological engineering field.

Background technology

Plant usually can be subjected to the stimulation of various environmental factors in process of growth; coerce the factor such as biologies such as the abiotic stress factor such as some arids, low temperature, high salt and infection process, mechanical wounding, insect pests, plant has been evolved in the process of these adverse circumstances of opposing and has formed a series of degeneration-resistant responsing reactions and defense mechanism.Transcription factor is in the status of center regulation and control in the process of these degeneration-resistant and defense responses of regulation and control plant.(Zinc finger protein ZnF) is one of transcription factor family bigger in the plant, the multiple regulation processes such as growth, light and degeneration-resistant reaction of the morphogenesis of involved in plant, gamete generation, embryo and floral organ to zinc finger protein.Data-speculative from database, C encodes in the Arabidopis thaliana ₂H ₂Type and C ₃The zinc-finger protein transcription factor gene of H type has 231 and 33 respectively.

In recent years, more and more evidences shows that zinc finger protein has participated in some and the correlated response of environment stresses such as arid, low temperature, high salt and disease and pest, and role also is diversified in these reactions, some is to activate Expression of Related Genes as activating son, some is to suppress some expression of gene as suppressing son, and also some can be combined into homology with other transcription factor or heterodimer plays a role in the regulation and control environment stress.At present, about the research of zinc finger protein mainly concentrates on Arabidopis thaliana, petunia, Common Snapdragon and paddy rice isotype species, the research that relevant zinc finger protein is carried out in the crop of genome complexity such as wheat is few.

Summary of the invention

The purpose of this invention is to provide a kind of plant stress-resistance zinc finger protein and encoding gene thereof.

Plant stress-resistance zinc finger protein provided by the present invention, name is called TaZnF (Triticum aestivum Zincfinger protein), derives from common wheat (Triticum aestivum L.), is following (a) or protein (b):

(a) protein of forming by the amino acid residue sequence of sequence in the sequence table 2;

(b) with the amino acid residue sequence of 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 stress resistance of plant by (a) deutero-protein.

Sequence 2 is made up of 165 amino-acid residues in the sequence table, as shown in Figure 7, contains two Zinc finger domains altogether, and wherein N end (sequence 2 from the 22nd of aminoterminal to 41 amino acids residues) coding A-20 zinc refers to (C ₂C ₂Type), C end (sequence 2 from the 106th of aminoterminal to 138 amino acids residues) coding AN-1 zinc refers to (C ₂H ₂Type).Among Fig. 7, underscore represents that respectively the A20 zinc of N end refers to and the AN-1 Zinc finger domain of C end that dash area represents to form the conserved amino acid of zinc fingers.

The replacement of described one or several amino-acid residue and/or disappearance and/or interpolation are meant replacement and/or disappearance and/or the interpolation of carrying out one to ten amino-acid residue outside above-mentioned two Zinc finger domains of sequence 2.

The encoding gene of above-mentioned plant stress-resistance zinc finger protein (TaZnF) also belongs to protection scope of the present invention.

Described plant stress-resistance zinc finger protein encoding gene, its nucleotide sequence are the proteinic polynucleotide of sequence 2 in the code sequence tabulation.

The encoding sequence (ORF) of described plant stress-resistance zinc finger protein encoding gene is the nucleotide sequence from the 1355th to 1852 deoxynucleotides compositions of 5 ' end of sequence 1 in the sequence table.

Described plant stress-resistance zinc finger protein encoding gene specifically can be following 1) or 2) gene:

1) its nucleotide sequence is the sequence 1 in the sequence table;

2) under stringent condition with 1) the dna sequence dna hybridization and the coding protein DNA molecule relevant that limit with stress resistance of plant.

Described stringent condition be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.

Sequence 1 in the sequence table is made up of 2147 deoxynucleotides.From the 1st to 1354 deoxynucleotides of 5 ' end is promoter region, is encoding sequence from the 1355th to 1852 deoxynucleotides of 5 ' end, from the 1853rd to 2147 deoxynucleotides of 5 ' end.

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

Arbitrary segmental primer is to also within protection scope of the present invention among the amplification TaZnF, 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 1:5 '-GCGGGATCACAAGCAGGAG-3 '; Primer 2: 5 '-GCCGCCGGAACATCAGCAT-3 '.

The carrier that utilization can guide foreign gene to express in plant, with TaZnF gene transfered plant cell of the present invention, or with the TaZnF gene overexpression, the resistance that plant shows as various abiotic stress adverse circumstances (arid, low temperature, high salt etc.) strengthens.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.The gene pairs of the present invention degeneration-resistant new variety that cultivate plants are significant.

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

Description of drawings

Fig. 1 be TaZnF because of the stress-inducing expression pattern

Fig. 2 is the Subcellular Localization result of the fusion rotein of TaZnF and GFP

Fig. 3 is the growth change curve under the Bacillus coli cells stress conditions that transforms pGEX-4T-1-TaZnF and pGEX-4T-1

Fig. 4 is that wild-type and transgenic line were handled one month in arid, and after the week that repeats to water, plant recovers the state photo of growth

Fig. 5 be wild-type and transfer-gen plant before subzero treatment, and subzero treatment is placed on and recovers the plant strain growth state photo of growth after 5 days and 10 days in the normal habitat

Fig. 6 be wild-type and transfer-gen plant before high salt is handled, and the plant strain growth state photo of handling back 7 days and 14 days

Fig. 7 is the aminoacid sequence of TaZnF

Embodiment

Following experimental technique if no special instructions, is ordinary method.

The clone of embodiment 1, TaZnF gene

(1) electronic cloning of wheat TaZnF gene order

Full length cDNA sequence with OSISAP1 gene in the paddy rice is a search sequence, login GenBank database (http://www.ncbi.nlm.nih.gov/BLAST/) then, est database is carried out the sequence homology comparison search (blastn), obtain 12 est sequences with the sequence height homologous wheat of paddy rice altogether, be respectively CD904159, CD934857, CA733262, BQ803525, BQ802055, BQ800826, CD870668, CD868128, BQ838331, AL825727, BJ322156, BJ320036 carries out sequence assembly with DNAMAN software; Utilize the result of splicing for the first time to carry out the sequence homology comparison second time and search (blastn), obtain the est sequence of 25 wheats, utilize DNAMAN software that these 25 est sequences are spliced with the result of comparison for the first time respectively, 1 est sequence (BJ27846) wherein splices the longest of back extension sequence with the result of the 1st splicing, then this splicing result is submitted to NCBI, utilizes blastx forecast function territory.

(2) cDNA of wheat TaZnF gene and non-translational region sequence clone

The sequences Design primer that obtains according to electronic cloning, cDNA with wheat breed agricultural university 3432 (Triticum aestivumL.) (available from Tongzhou District, Beijing City seeds company) blade is a template, carry out pcr amplification, the PCR product obtains TaZnF gene ORF section through after the sequence verification.The non-translational region sequence of utilizing 3 ' RACE to obtain 3 ' end of TaZnF gene is 295bp, has 19 polyA tails.Utilize Genome walker technology to obtain the promoter region 1354bp of 5 ' end, complete sequence information is seen the SEQ ID № in the sequential analysis table: 1, simultaneously the cis acting composition of promoter region is analyzed; The protein sequence of the ORF sequence encoding of TaZnF such as the SEQ ID № in the sequence table: 2, contain two Zinc finger domains altogether, wherein N end (sequence 2 from the 22nd of aminoterminal to 41 amino acids residues) coding A-20 zinc refers to (C ₂C ₂Type), C end (sequence 2 from the 106th of aminoterminal to 138 amino acids residues) coding AN-1 zinc refers to (C ₂H ₂Type), and the TaZnF gene be a zinc finger protein gene that does not contain intron.

The stress-inducing expression pattern of embodiment 2, TaZnF gene

Handle wheat seedling respectively with 25%PEG-6000,250mM NaCl, 4 ℃ of low temperature and 100uM ABA, and get the wheat root of different treatment time, after extracting RNA reverse transcription cDNA, use primer 1:5 '-GCGGGATCACAAGCAGGAG-3 ' and primer 2: 5 '-GCCGCCGGAACATCAGCAT-3 ', adopt real-time fluorescence quantitative PCR to detect the spatial and temporal expression feature of TaZnF gene.The concrete grammar of wherein coercing processing is as follows:

Experiment material is common wheat kind agricultural university 3432 (Triticum aestivum L.), and the clorox of seed-coat sterilization employing 5% was handled 10 minutes, behind distilled water flushing 4 times, places 4 ℃ of refrigerators secretly to cultivate a week.The bottom of culturing bottle is put the wetted cotton in advance well, and the seed of wheat is moved into culturing bottle, places culturing room (25 ℃ of temperature, relative humidity 60%-70%, light intensity 7000-8000Lux) water planting to support.After one heart stage, coerce processing at wheat growth to two leaf:

High salt is handled: adding NaCl solution to final concentration in culturing bottle is 250mM.

PEG handles: adding PEG-6000 solution to final concentration in culturing bottle is 25% (quality percentage composition).

ABA handles: ABA is dissolved in makes the 10mM stock solution among the DMSO, join then in the culturing bottle to final concentration be 100uM.

After adding above-mentioned solution, handling the root system of choosing wheat in 0.5,1,2,12,24 hour respectively, place liquid nitrogen rapidly after blotting residual solution with thieving paper, in-80 ℃ of refrigerators, preserve, stand-by.With untreated wheat root (promptly only being grown in the water), organize in contrast simultaneously.

Subzero treatment is that the seedling with wheat places 4 ℃ of low temperature and irradiance incubators, and the time and the method for drawing material of processing are the same, and control group is temperature (25 ℃) and the illumination condition (7000-8000Lux) in normal culturing room.

The result of real-time quantitative fluorescence PCR as shown in Figure 1, the TaZnF gene be subjected to PEG (25%), high salt (NaCl, 250mM), low temperature (4 ℃) and ABA (100 μ M) handle that corresponding response is all arranged.Processing for PEG (25%), the initial stage (0.5 hour) of handling, expression of gene rapidly increases to highest level, then along with the prolongation in treatment time, the expression of gene amount begins to descend, to later stage (12 hours), gene expression amount remains essentially in (A among Fig. 1) on the metastable level.After plant hormone ABA handles (100uM), expression of gene is elevated to (2 hours) after the highest level gradually, slowly descends (B among Fig. 1).During subzero treatment (4 ℃), in initially treated stage (0.5 hour), expression of gene rapidly increases to highest level, descends fast again then, to later stage (12 hours) slightly rising again, maintains then (C among Fig. 1) on the metastable level.(NaCl, 250mM), genetic expression began to descend along with the increase in treatment time slowly is elevated to the highest (12 hours) then, up to later stage (24 hours) expression amount even will be lower than background level (D among Fig. 1) when high salt was handled.Among Fig. 1, the time that X-coordinate is coerced processing was respectively 0.5,1,2,12,24 hour, and ordinate zou is illustrated in relative expression's level of TaZnF gene under the different treatment condition.

The Subcellular Localization of embodiment 3, TaZnF gene

Be the site of action of checking TaZnF gene in cell, pEGAD (Sean R.Cutler is inserted in the coding region of TaZnF, David W.Ehrhardt, Joel S.Griffitts, and Chris R.SomervilleRandom GFP::cDNA fusions enable visualization of subcellular structuresin cells of Arabidopsis at a high frequency, PNAS, 2000, (97) obtain containing the recombinant expression vector pEGAD-TaZnF of GFP-TaZnF fusion gene between 35S promoter 3718-3723) and the green fluorescent protein GFP gene, determining TaZnF albumen in intracellular location situation by the GFP expressive site, is contrast with the pEGAD expression vector simultaneously.Change above-mentioned two kinds of carriers over to onion epidermis cell through particle bombardment respectively, the result shows that the green fluorescence of the onion epidermis that changes pEGAD-TaZnF is distributed in the nucleus (A among Fig. 2～C), and the green fluorescence that the GFP albumen of control group sends is evenly distributed in (D among Fig. 2) in the whole cell.The albumen that shows the TaZnF genes encoding is a nucleoprotein.Among Fig. 2, A, B, C represent that respectively TaZnF albumen is positioned in the nucleus of onion epidermis cell, and A is the details in a play not acted out on stage, but told through dialogues photo, and B is the light field photo, and C is the photo after A and the B stack.D be empty carrier pEGAD in the location of onion epidermis cell situation, be evenly dispersed in the whole cell.

Embodiment 4, TaZnF expression of gene strengthen the resistance of prokaryotic organism Bacillus coli cells

In order to verify TaZnF impermeabilisation, high salt tolerance and cryophylactic effect in prokaryotic organism, made up prokaryotic expression carrier pGEX4T-1-TaZnF.The construction process of pGEX4T-1-TaZnF is as follows: the cDNA with wheat breed agricultural university 3432 (Triticum aestivum L.) blade is a template, utilize primer 2: 5 '-CGGAATTCATGGCGCAGCGGGATCACAG-3 ' and 5 '-CCCAAGCTTTCAGAACCTGACGATCTTGGC-3 ', pcr amplification obtain TaZnF encoding sequence (in the sequence table sequence 1 from 5 ' the 1355th to 1852 deoxynucleotides of end), the TaZnF fragment that obtains is inserted between the restricted type restriction endonuclease EcoRI and HindIII restriction enzyme site of pGEX-4T-1 (available from Pharmacia company), obtained containing the segmental recombinant expression vector pGEX4T-1-TaZnF in TaZnF coding region.

With pGEX4T-1-TaZnF transformed into escherichia coli (E.coli) BL21 (DE3) cell, use pGEX-4T-1 (available from Pharmacia company) transformed into escherichia coli cell (E.coli) BL21 (DE3) simultaneously, in contrast.It is 0.6 o'clock that the intestinal bacteria that transform are cultured to OD600 value LB substratum, 37 ℃, and adding 0.1mmol/L IPTG induces.IPTG carries out following any processing to nutrient solution respectively after inducing 2hr: 1) add 1M N.F,USP MANNITOL in nutrient solution; 2) in nutrient solution, add 0.3M LiCl solution; 3) bacterium liquid is placed 10 ℃ low temperature shaking culture case to cultivate to take out the shaking culture case that places 37 ℃ behind the 24hr recover growth.Detect respectively that the Bacillus coli cells that Bacillus coli cells that pGEX4T-1-TaZnF transforms and pGEX-4T-1 transform oozes at height, the growing state in high salt and the low temperature environment.Experimental result is shown in A-C among Fig. 3: A was illustrated in after the Mannitol that adds 1M among Fig. 3, surveyed the OD600 value of bacterium liquid every two hours; B was illustrated in after the LiCl that adds 300mM among Fig. 3, surveyed bacterium liquid OD600 value every two hours; C was illustrated under 10 ℃ of conditions and cultivates after 24 hours among Fig. 3, moves on in 37 ℃ the environment to cultivate, and surveyed the OD600 value of bacterium liquid every 1 hour.

A shows in the adding 1mol/L N.F,USP MANNITOL 2hr among Fig. 3, the intestinal bacteria continued growth that is transformed, and the speed of growth slows down behind the 2hr, and the Bacillus coli cells that part is transformed is influenced by N.F,USP MANNITOL, and is dead gradually; Compare with the Escherichia coli bacteria liquid absorption value that pGEX4T-1-TaZnF transforms, the lowering speed of contrast bacterium liquid absorption value is fast; Most of bacterial cell begins to recover growth behind the 6hr, but the Bacillus coli cells that pGEX4T-1-TaZnF transforms recovers the speed of growth also faster than the cell that transforms empty carrier pGEX4T-1.Show that TaZnF has improved the ability of Bacillus coli cells impermeabilisation.

B shows in the adding 0.3M LiCl 2hr among Fig. 3, two class cells are in the growth conditions of a stagnation basically, behind the 2hr, the cell that transforms pGEX4T-1-TaZnF begins to recover at leisure growth, and the cell of conversion empty carrier pGEX-4T-1 is in the state of stagnating growth basically.Show that TaZnF has strengthened Bacillus coli cells for salt ion (Li ⁺) suffertibility.

C shows and places bacterium liquid 10 ℃ low temperature shaking culture case to cultivate 24hr among Fig. 3, handles initial stage part Bacillus coli cells growth, then stays cool basically.Take out the shaking culture case that places 37 ℃ behind the 24hr and recover growth, the result shows that the cellular-restoring speed of growth that transforms pGEX4T-1-TaZnF is than the high cell growth speed that transforms empty carrier pGEX-4T-1.It is faster than the Bacillus coli cells growth that contains empty carrier to show that the Bacillus coli cells that transforms pGEX4T-1-TaZnF recovers the ability of growth behind low temperature stress.Show that TaZnF has strengthened Bacillus coli cells for cryogenic resistibility.

(LB substratum in normal habitat, 37 ℃), transform the Bacillus coli cells of pGEX4T-1-TaZnF and the growth conditions and the speed of growth basically identical of control cells, there is not very big difference, but ooze at height, under high salt or the low temperature stress, the Bacillus coli cells that transforms pGEX4T-1-TaZnF generally shows the stronger energy for growth and the speed of growth faster than cellular control unit.

Embodiment 5, wild-type and transgenic plant are identified at different phenotype and physiology of coercing under the treatment condition

1, the acquisition of transfer-gen plant

(1) structure of pCAMBIA-35S-TaZnF expression vector

CDNA with wheat breed agricultural university 3432 (Triticum aestivum L.) blade is a template, utilize primer 3:5 '-GCTCTAGAATGGCGCAGCGGGATCACA-3 ' and 5 '-GGGGTACCTCAGAACCTGACGAGCTTG-3 ', pcr amplification obtain TaZnF encoding sequence (in the sequence table sequence 1 from 5 ' the 1355th to 1852 deoxynucleotides of end), with the TaZnF fragment that obtains after restriction enzyme XbaI and KpnI digestion, insert between the restricted type restriction endonuclease XbaI and KpnI restriction enzyme site of plasmid pCAMBIA-1300 (available from CAMBIA company), obtain containing the segmental expression vector pCAMBIA-TaZnF in TaZnF coding region; With restriction enzyme Hind III and Xba I digested plasmid pBI121 (available from Clontech company), reclaim the CaMV35S fragment, and insert between the restricted type restriction endonuclease Hind III and XbaI enzyme cutting site of plasmid pCAMBIA-TaZnF, obtain containing the segmental overexpression vector pCAMBIA-35S-TaZnF in CaMV35S promotor and TaZnF coding region.

Transform agrobacterium tumefaciens lba4404 with pCAMBIA-35S-TaZnF, utilize Agrobacterium to infect method and transform the environmental Arabidopis thaliana (Arabidopsis Biological Resource Center) of Columbia, obtained (the T of 30 commentaries on classics pCAMBIA-35S-TaZnF strain systems (be called for short and change TaZnF gene strain system) altogether through 50ug/l Totomycin and PCR screening ₃Generation).With wild-type (WT) and T ₃In generation, changes TaZnF gene plant seed and is tiled on the MS substratum, and behind 4 ℃ of following vernalization 5d, (light intensity is 30-40umol.m for 22 ℃ of constant temperature, 24hr illumination in the immigration growth case ^-2s ^-1) cultivate after ten days and move in the soil.With the wild-type contrast, changeing TaZnF gene strain system does not have obvious phenotypes to change.Choose 30 5 strain systems (#2, #4, #13, #14, #28) that change in the TaZnF gene strain system and carry out the transfer-gen plant resistance evaluation of following step 2.

Wherein, the first-generation transfer-gen plant that obtains is T ₁Generation, T ₁The seed of tying for transfer-gen plant and be T by the plant that this seed grows up to ₂In generation, the rest may be inferred, T ₃Expression the 3rd generation of transfer-gen plant.

2, the resistance of transfer-gen plant is identified

(1) expresses the transgenic arabidopsis drought resistance analysis of TaZnF excessively

Start the expression of TaZnF gene in transfer-gen plant by 35S promoter, detect the effect of TaZnF gene in drought stress.Concrete grammar is as follows: at first with WT and T ₃In generation, changeed the vernalization 5 days in culture dish of TaZnF gene strain system (#2, #4, #13, #14, #28) seed kind, moves in the incubator (light intensity is 30-40umol.m-2s-1 for 22 ℃ of constant temperature, illumination in 24 hours) and cultivate and moved in the soil regrowth after ten days 14 days.Before arid is handled, select onesizely, it is that plant (each 81 strain) places same pallet that the WT of growth conditions unanimity and #2, #4, #13, #14, #28 change the strain of TaZnF gene, and 3 repetitions are set; Stopped to water 30 days and carry out arid and handle, repeated then to water 7 days, the survival rate (seeing Table 1) of statistics strain system, from the statistics of survival rate, the survival rate of wild-type is 22.38%, and the survival rate of changeing TaZnF gene strain system is respectively 53.73%, 65.15%, 76.47%, 89.87% and 100%, the result that T-detects shows: the survival rate of changeing the strain of TaZnF gene and be #2, #4, #14, #28 is compared with wild-type all significant difference (P＜0.05).From observing in appearance, the phenotype of changeing TaZnF gene strain system and wild-type is consistent.WT and the strain of commentaries on classics TaZnF gene tie up to after the arid processing, the blade of plant is dehydration wilting gradually all, the speed of the plant dehydration of commentaries on classics TaZnF gene strain system is slower than WT plant slightly, but difference is not obvious, but after arid, repeat to water after a week, most of WT plant leaf begins flavescence, turns white, and dies gradually, and change the strain of TaZnF gene is that most of plant can recover normal level of growth, and finishes whole life cycle (Fig. 4).Above result proves that the survival rate of changeing TaZnF gene strain system is generally greater than WT, and the ability of recovering growth after repeating to water is far longer than WT, illustrates to cross in Arabidopis thaliana and expresses the drought-resistant ability that the TaZnF gene has improved transfer-gen plant significantly.

Table 1: wild-type (WT) and the strain of commentaries on classics TaZnF gene tie up to the survival rate under the drought condition

Strain system	Survival rate
		WT #
2 #4 #13 #14 #28	22.38％ 84.31％* 100％* 53.73％ 65.15％* 89.87％*

Annotate: * represents significant difference (P＜0.05)

(2) anti-low temperature is identified

1. the survival rate under the cold condition is measured

WT and T ₃Cultivate the same early stage of changeing TaZnF gene strain system (#2, #4, #13, #14, #28) plant in generation, before subzero treatment, select onesize, the WT of growth conditions unanimity and T ₃In generation, changes TaZnF gene strain system (#2, #4, #13, #14, #28) (each 81 strain) and places same pallet, and 3 repetitions are set; Plant was placed-10 ℃ of incubators 4 hours, took out in the incubator be placed on-5 ℃ subzero treatment 30 hours, take out then and place under the normal culture condition (22 ℃) growth after 10 days, add up the survival rate (table 2) of whole strain system.From observing in appearance, the strain of commentaries on classics TaZnF gene is that the phenotype of plant and wild-type is consistent.WT and the strain of commentaries on classics TaZnF gene tie up to after the subzero treatment; the blade of plant all has frostbite in various degree; WT and the strain of commentaries on classics TaZnF gene are that difference is not obvious; but in home, cultivate after 5 days; most WT plant leaf begins to bleach; and die gradually, and the strain of most of commentaries on classics TaZnF gene is that plant can recover normal level of growth basically, and finish whole life cycle (Fig. 5).The result that T-detects shows: change the strain of TaZnF gene and tie up to survival rate that low temperature stress recovers later on to grow generally greater than WT, and the survival rate that three strain systems (#4, #13 and #28) are arranged is compared with wild-type all significant difference (P＜0.05), a strain system (#14) and wild-type and the difference (P＜0.01) that is in a ratio of utmost point significance, and the ability that growth recovers in TaZnF gene strain system behind the low temperature stress illustrates and cross the cryophylactic ability that the TaZnF gene has improved transfer-gen plant significantly of expressing in Arabidopis thaliana also greater than WT.

2. low temperature stress is handled the mensuration of excised leaf specific conductivity down

The cytolemma of plant plays an important role to microenvironment and the normal metabolism of keeping cell, under normal circumstances, cytolemma has the saturating sexuality of selection to material, when being subjected to adverse circumstances such as low temperature when plant and influencing, cytolemma can be destroyed, membrane permeability increases, thereby intracellular ionogen is exosmosed, so that the specific conductivity of vegetable cell vat liquor increases.The variation of specific conductivity is relevant with the resistance of plant under identical stress conditions, and promptly the degree that is hurt of the strain cytolemma of strong stress resistance is little, and cell membrane stability (Cell membrane stability, CMS) strong.

To WT and T ₃Generation is changeed TaZnF gene strain system (#2, #4, #13, #14, #28) plant (respectively 5 strains of every strain system), and the specific conductivity of excised leaf is measured when low temperature stress, CMS=[1-(T1/T2)]/[1-(C1/C2)].The mensuration of specific conductivity adopts following method: 1) laying diameter with punch tool from transgenosis and wild-type plant the 5th and the 6th leaf is that (each genotype is done five repetitions for 0.5 centimetre disk, take from the different plant of five strains respectively, two leaves are taken off in every strain, a slice leaf is as treatment group, and a slice leaf is organized in contrast), use deionized water rinsing 2-3 time, after thieving paper blots, place the centrifuge tube of 10ml, add deionized water 5ml, placed under-10 ℃ of conditions the dark place reason 24 hours.2) mensuration of the specific conductivity of the blade of control group is directly to add to remove dried up 5ml in centrifuge tube, after making whole blade all be immersed in the water, be placed in 4 ℃ of refrigerators after 24 hours, at room temperature balance 1-2 hour, measure the specific conductivity of solution in each centrifuge tube then with conductivitimeter, the reading of the specific conductivity of writing down is C1; Then, at 15Psi, sterilization is 15 minutes under 121 ℃ of conditions, measures the specific conductivity of solution this moment again with conductivitimeter, is designated as C2.3) mensuration of the specific conductivity of treatment group is undertaken by as above operating, and the specific conductivity before and after the autoclaving is T1 and T2.

The result shows that the CMS value of wild-type plant leaf after subzero treatment is 27.04%, and the CMS value of changeing TaZnF gene strain system all is respectively 53.35%, 54.94%, 57.78%, 64.72% and 73.77%, and 3 CMS values of changeing TaZnF gene strain system (#2, #4 and #13) have been compared significant difference with wild-type, 2 are changeed TaZnF gene strain system (#14 and #28) utmost point significant difference (table 2) are arranged, explanation is under the low temperature stress condition, and the cell membrane stability of changeing TaZnF gene strain system generally will be higher than wild-type.

Table 2: wild-type (WT) and change the strain of TaZnF gene and tie up to survival rate under the cold condition and excised leaf

The CMS pH-value determination pH

Strain system	Survival rate	CMS
			WT #
2 #4 #13	64.71％ 85.71％* 100％* 91.18％*	27.04％ 57.78％* 54.94％* 53.35％*

#14 #28

96.42％** 92.86％*

64.72％** 73.77％**

Annotate: * and * * represent significant difference (P＜0.05) and utmost point significant difference (P＜0.01) respectively

(3) salt resistance is identified

1. the survival rate under high-salt stress is handled is measured

WT and T ₃Cultivate the same early stage of changeing TaZnF gene strain system (#2, #4, #13, #14, #28) plant in generation, before high salt is handled, select onesize, the WT of growth conditions unanimity and T ₃In generation, changes TaZnF gene strain system (#2, #4, #13, #14, #28) (each 81 strain) and places same pallet, and 3 repetitions are set, and cultivates after 5 days, in pallet, execute the NaCl solution that waters 400mM, watered once every five days, water 500ml at every turn, the survival rate (table 3) of the whole strain of statistics system after 14 days.Wild-type is consistent with the phenotype of changeing TaZnF gene strain system.After salts solution is handled 7 days, wild-type and the strain of commentaries on classics TaZnF gene are that indivedual blades of plant begin to bleach, handle after 14 days, the strain of most of commentaries on classics TaZnF gene is that the blade of plant part all bleaches, but the WT blade degree of turning white is more serious relatively than changeing TaZnF gene strain system by contrast, and the ratio that is hurt is also wanted big (Fig. 6).Above result shows that the survival rate that the strain of commentaries on classics TaZnF gene ties up to after high salt is handled is a bit larger tham WT, and the speed that blade bleaches is also slow than WT, illustrates that the ability of crossing the high salt tolerance of expressing TaZnF gene pairs transfer-gen plant in Arabidopis thaliana slightly improves.

2. the mensuration of high-salt stress condition lower blade chlorophyll content

According to document " plant physiology and biochemistry experimental principle and technology " (Li Hesheng etc., 2000, Beijing, Higher Education Publishing House, 134-137) method of Miao Shuing is to WT and T ₃In generation, changeed the TaZnF gene strain (#2 of system, #4, #13, #14, #28) (every strain system each 5 strains) chlorophyll content of blade of 7 days behind high-salt stress is measured, the result shows that the chlorophyllous content of WT after high salt is handled is 1.30mg/g, and the chlorophyllous content of transgenic line is respectively 1.48mg/g, 1.79mg/g, 1.78mg/g, 1.94mg/g and 1.95mg/g above (table 3), and #2, #13, #14 and #28 transgenic line are after height is coerced, chlorophyll content is compared with wild-type all significant difference, these results show, the strain of commentaries on classics TaZnF gene ties up to and has the chlorophyll content higher than WT under the high-salt stress, and also TaZnF gene strain system is changeed in explanation has the stronger ability that salt ion is coerced of standing than WT.

Table 3: wild-type (WT) and the strain of commentaries on classics TaZnF gene tie up to survival rate and the chlorophyll measuring under the high salt condition

Strain system	Survival rate	Chlorophyll content (mg/g)
			WT #2 #4 #13	80.65％ 97.22％* 86.11％ 88.89％	1.30 1.95* 1.48 1.79*

#14 #28

91.67％* 95.56％*

1.94* 1.78*

Annotate: * represents significant difference (P＜0.05)

The expression of The above results explanation wheat TaZnF gene in Arabidopis thaliana improved that transfer-gen plant is drought-resistant, the ability of low temperature and high salt, and the growth and development process of transfer-gen plant is not affected when improving resistance.

Sequence table

<160>2

<210>1

<211>2147

<212>DNA

＜213〉Triticum wheat (Triticum aestivum L.)

<400>1

ccgtgggatt agccgtccgc ggaaatgata cggccgccga atttctgctc gccgcgatgc 60

gaccttggct tcgtgcggct gctgtctccg ggccggccgg gagaatctgc tggattgctt 120

acccagttcg ctctgctgtt ggcctgtctg ttcctgattt gccgccgcgc cctaccgacc 180

gaattcagca cctgaagact agctaatgga agtttttttt tccttaaaaa aaagttagca 240

gcagctagcc gggatgcgtg ggcagaatgg cccggccgtg cggttgggac tatgcacgag 300

tttgccgtgt cgacactcaa atcggatcgt aattgagtat tcccgtctag cgatattttc 360

tactagtact attagcgata agataaacga atgggcgaga ggtgaagagg ggagcagccg 420

tgctaatccg gacagctcag aggcggacaa ggacggggcg ccgatccagc ttcccttaat 480

tttatcccga ccctggccgc aacgtgcggg ctatggtatg gtggtacgta cgcacggaag 540

aagccgcgac gacgccttcc aagttccaac tcgcaggccc cgacaagccg acgcgcccga 600

tggtttggcc cggtggtttc gtcccaaatt cccagccatc ttctcgctcg ctttcggccg 660

ccatactagt cagcttcggg cggcggctac ttgctccccc acacggcgcg gccggccgtg 720

cgggtccaac cagcacacgg cacacacggc ccgtccgtgc ccacgaagcg ccacctgacc 780

atcctacccc tccttcgcac ctacgcgacg tccgtgcgtg ctccggcgcc cctctggatc 840

cagcccagtt gacccttctc cccaccaccc gcggccacgg gcgtcgcacg tcgctcacgt 900

ttcttccatg gagggcctaa acgggcgtgc ctttaccggc ctgcagcaca gccgtcgctt 960

ttcgggggcg gccactgacc gaaacggagg gcagcgtcgc ccatttacgc gagcccccgg 1020

gtgacgcgag gccaaaaccg gaaatcccaa cccaagacgc ggagggagac ggcagctggg 1080

caggtcgtta tcgcccctcc tccagggagc gcgtccccgt ccgtctcccg cctagccctc 1140

ggcaccgata aaaagggccc cggtctcccc tccattcccc acccacgccc atccaattct 1200

cctctcggcc tctcctcctc tcctcgcatc ggcctcccga tccctcacag accacccgcg 1260

gcgcgcacac gcccggacct cctaccggcc ggcaccggtt caaccaacaa accagcccag 1320

gcacctccgg ccgcgcgtgc gtgtttgaca taccatggcg cagcgggatc acaagcagga 1380

ggagcccacg gagctgcggg cgccggagat cacgctctgc gccaacagct gcggcttccc 1440

gggcaacccg gccacgcaga acctctgcca gaactgcttc ttggccggcc cggcgtccac 1500

gtcgccgtct tcctcctcct cctcctcctc ttctctgccg ggcgtgtccg cgccgacccc 1560

cgtcatcgac aggccgaggc cggcgccgtt ggaggcggag ctggcacgcc ccgccgtcga 1620

ccttgctccg gcgacggagg cgaagccggc gaggacgtcg gtgaaccggt gctccagctg 1680

ccggaagcgc gtggggctga cggggttccg gtgccggtgc ggcgacatgt tctgcggcga 1740

gcaccggtac tcggaccggc acgggtgcag ctacgactac aaggccgccg ccagggacgc 1800

catcgccagg gacaaccccg tcgtgcgcgc cgccaagctc gtcaggttct gaagctgaaa 1860

ccaaggggaa agaagaattc agaattcagc cagaggcaaa gcaaagaatc atctcatcga 1920

ggcagtgggc cgccgtgcga gccgctagta ttatgctgtt gttctccgga gtgcttccct 1980

ttttaattaa aaaaatcttt atttattgga aggaaaaaaa tgggaggtaa agatgtggat 2040

gaaaaggaaa gaatagcgtg tgtgatgtaa tatcaacggg tggttgaggc ggaggagcaa 2100

gcaatgaaat ggattggatg tgtatgtcaa aaaaaaaaaa aaaaaaa 2147

<210>2

<211>165

<212>PRT

＜213〉Triticum wheat (Triticum aestivum L.)

<400>2

Met Ala Gln Arg Asp His Lys Gln Glu Glu Pro Thr Glu Leu Arg Ala

1 5 10 15

Pro Glu Ile Thr Leu Cys Ala Asn Ser Cys Gly Phe Pro Gly Asn Pro

20 25 30

Ala Thr Gln Asn Leu Cys Gln Asn Cys Phe Leu Ala Gly Pro Ala Ser

35 40 45

Thr Ser Pro Ser Ser Ser Ser Ser Ser Ser Ser Ser Leu Pro Gly Val

50 55 60

Ser Ala Pro Thr Pro Val Ile Asp Arg Pro Arg Pro Ala Pro Leu Glu

65 70 75 80

Ala Glu Leu Ala Arg Pro Ala Val Asp Leu Ala Pro Ala Thr Glu Ala

85 90 95

Lys Pro Ala Arg Thr Ser Val Asn Arg Cys Ser Ser Cys Arg Lys Arg

100 105 110

Val Gly Leu Thr Gly Phe Arg Cys Arg Cys Gly Asp Met Phe Cys Gly

115 120 125

Glu His Arg Tyr Ser Asp Arg His Gly Cys Ser Tyr Asp Tyr Lys Ala

130 135 140

Ala Ala Arg Asp Ala Ile Ala Arg Asp Asn Pro Val Val Arg Ala Ala

145 150 155 160

Lys Leu Val Arg Phe

165

Claims (10)

1, plant stress-resistance zinc finger protein is following (a) or protein (b):

(a) protein of forming by the amino acid residue sequence of sequence in the sequence table 2;

(b) with the amino acid residue sequence of 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 stress resistance of plant by (a) deutero-protein.

2, plant stress-resistance zinc finger protein according to claim 1 is characterized in that: the protein that described plant stress-resistance zinc finger protein is made up of the amino acid residue sequence of sequence in the sequence table 2.

3, the encoding gene of claim 1 or 2 described plant stress-resistance zinc finger proteins.

4, gene according to claim 3 is characterized in that: described plant stress-resistance zinc finger protein encoding gene, its nucleotide sequence are the proteinic polynucleotide of sequence 2 in the code sequence tabulation.

5, gene according to claim 4 is characterized in that: the encoding sequence of described plant stress-resistance zinc finger protein encoding gene is the nucleotide sequence from the 1355th to 1852 deoxynucleotides compositions of 5 ' end of sequence 1 in the sequence table.

6, according to claim 3,4 or 5 described genes, it is characterized in that: described plant stress-resistance zinc finger protein encoding gene is following 1) or 2) gene:

1) its nucleotide sequence is the sequence 1 in the sequence table;

2) under stringent condition with 1) the dna sequence dna hybridization and the coding protein DNA molecule relevant that limit with stress resistance of plant.

7, the expression vector that contains arbitrary described plant stress-resistance zinc finger protein encoding gene in the claim 3 to 6.

8, the transgenic cell line that contains arbitrary described plant stress-resistance zinc finger protein encoding gene in the claim 3 to 6.

9, the transformed host bacterium that contains arbitrary described plant stress-resistance zinc finger protein encoding gene in the claim 3 to 6.

10, the application of arbitrary described plant stress-resistance zinc finger protein encoding gene in cultivating the adversity resistant plant kind in the claim 3 to 6.

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