CN102268443A - Application of corn WRKY gene in enhancing plant stress tolerance - Google Patents

Abstract

The invention discloses application of a corn WRKY gene in enhancing plant stress tolerance. The amino acid sequence of the corn WRKY gene is SEQ ID NO:1 in the sequence table; and the amino acid sequence SEQ ID NO:1 in the sequence table is subjected to substitution and/or deletion and/or addition of one or more amino acids, and has the functions of a plant WRKY transcription factor. The T1 generation plant stress tolerance experiment of the over-expression transgenic plant system of the corn WRKY transcription factor proves that the transgenic survival rate under stress conditions is basically higher than 60%, which indicates that the corn WRKY gene can obviously enhance the tolerance to low temperature, high salt and drought stress. The protein and coding gene thereof have important theoretical and practical meanings for researching plant stress tolerance, enhancing plant stress tolerance and improving related characters, can perform vital functions on improving plant stress tolerance gene engineering, and has wide application prospects.

Description

A kind of corn WRKY gene is in the application that improves on the plant stress tolerance energy

Technical field

The invention belongs to molecular biology of plants and molecular genetics field, specifically, relate to the application in cultivating the resistance of reverse plant of a corn WRKY transcription factor and encoding gene thereof.

Background technology

Zea Gramineae Zea.Whole world corn seeding area is only second to wheat, paddy rice and occupies the 3rd.Corn is not only important crops, also is simultaneously the industrial raw material and the first-selected fodder crop of developing animal husbandry, and occupies an important position in national economy.Existing kind can not satisfy the needs of this zone Maize Production, shows that mainly the resistance of promoting the back kind weakens year by year.Therefore excavate and create strong stress resistance, high yield, high-quality specialized corn new germ plasm and seed selection new variety become the key point of realization Maize Production sustainable development the sixth of the twelve Earthly Branches.

Transcription factor is also referred to as trans-acting factor, is can be conjugated protein with the interactional DNA of cis-acting elements generation specificity in the eukaryotic gene promoter region, the gene of being controlled is risen to transcribe activate or restraining effect.Typical plant transcription factor has DNA in conjunction with territory, transcriptional regulatory domain, oligomerization site and nuclear localization signal.The multiple vital movement of transcription factor wide participation biology, thereby transcription factor also has significant diversity, they are in growth, growth and the morphogenesis of higher plant, and play important regulation in the environment reactions such as biological and abiotic stress.

The WRKY transcription factor family is exactly the class in the numerous plant transcription factor family, mainly is present in the higher plant, comprises that in some unicellular lower eukaryotes low the grade in algae and the protozoon also has a small amount of discovery.It contains one or two conservative WRKY structural domain, and the WRKY structural domain contains about 60 amino-acid residues and promptly connects a zinc fingers after WRKYGQK seven amino acid residue core sequence.The WRKY territory can combine target gene expression by the special W-box with the target gene promoters district.

At present by in the plants such as Arabidopis thaliana, paddy rice to WRKY family member's functional analysis, we find that WRKY family member function is extensive, the not only multiple biological adverse circumstance factor of WRKY family member gene comprises inducing and regulating growth of plants, morphogenesis and metabolic regulation and signal conduction thereof of virus, bacterium, fungi etc., induced by the multiple abiotic stress factor such as low temperature, high temperature, arid, high salt, light (radiation), high humidity and damage etc. and participates in the abiotic stress reaction.The research of the WRKY family gene in the corn does not also seldom have direct data and the relevant function of experimental evidence proof but up to the present.So corn clone WRKY family member gene obtains transfer-gen plant by making up the overexpression carrier, sees whether it responds to stress conditions, and whether the gene of being studied can improve the ability that plant is coerced to external world effectively.

Summary of the invention

The objective of the invention is corn WRKY family member is carried out functional study, thereby obtain corn WRKY transcription factor and the gene thereof relevant, to be used to improve the resistance of reverse energy of plant with resistance of reverse.

The present invention utilizes the gene order in MAIZEGENOME and the ncbi database, and corn WRKY family member is carried out careful comparison and analysis, screens, cloned a gene ZmWRKY33, and prove that it has the function that strengthens rice stress-tolerance.

Concrete technical scheme of the present invention is as follows:

A kind of corn WRKYGene is in the application that improves on the plant stress tolerance energy, and is described WRKYThe genes encoding following proteins: aminoacid sequence is SEQ ID N0:1 in the sequence table.

With described WRKYGene transfered plant cell, tissue or organ will be cultivated into plant by plant transformed cell, tissue or organ again, obtain the transfer-gen plant that resistance of reverse improves.

Described WRKYGene is one of following nucleotide sequence: SEQ ID N0:2 or 3 dna sequence dna in the sequence table; Perhaps for having 90% above homology and the proteic dna sequence dna of coding identical function with SEQ ID N0:2 or 3 sequences.

Described WRKYGene imports vegetable cell, tissue or organ by plant expression vector.

Described plant is dicotyledons or monocotyledons.

The invention provides a kind of corn WRKYGene is in the application that improves on the plant stress tolerance energy.Experiment showed, that the gene transformation paddy rice that carries out an invention can improve the tolerance of paddy rice to arid, high salt and low temperature environment stress, and to paddy rice normal growth and not significantly influence of economic characters.The T1 of the mistake express transgenic strain of corn WRKY33 transcription factor gene system shows that for the experiment of plant resistance of reverse the transgenosis survival rate more than 60%, illustrates corn substantially under adverse circumstance among the present invention WRKY33Gene can significantly improve the resistance of transgenic line to low temperature, high salt and drought stress.Albumen of the present invention and encoding gene thereof are for the anti-contrary Study on Mechanism of plant, and improve the resistance of reverse of plant and the improvement of associated shape has important theory and practical significance, to in the anti-contrary genetically engineered improvement of plant (particularly cereal crop), play a significant role, have a extensive future.

Description of drawings

Fig. 1 is the Subcellular Localization photo of ZmWRKY.

A and B are PRTL2-mGFP carrier transient expression photo, and C and D are PRTL2-ZmWRKY-mGFP carrier transient expression photo.A and C are the onion cell under the no excitation light irradiation, and B and D are the onion cell under the irradiation of 445-490nm excitation light source.

Fig. 2 is through the ZmWRKY of drought stress T1 transfer-gen plant and wild-type plant photo.

ZmWRKY T1 transfer-gen plant and the wild-type plant photo of Fig. 3 for coercing through salts solution.

Fig. 4 is through the ZmWRKY of low temperature stress T1 transfer-gen plant and wild-type plant photo.

Among Fig. 2,3,4 A for respectively coerce handle before the photo of seedling growth conditions, B for respectively coerce restore normal growth after the processing before the photo of seedling growth conditions, the photo of seedling growth conditions after C restore normal growth 7 days, D for statistics each

Transgenic line and the survival rate of contrast wild-type plant after coercing processing.

Embodiment

Below in conjunction with specific embodiment the present invention is described in further details.

Method therefor is ordinary method if no special instructions among the following embodiment, the primer is synthetic by Shanghai Jierui Biology Engineering Co., Ltd, order-checking is undertaken by Invitrogen company, the PCR test kit, connect endonuclease in test kit and the vector construction process all available from from precious biotechnology company limited, the reverse transcription test kit is purchased the company in Promega, plasmid extraction kit and glue reclaim test kit and all purchase the Bioisystech Co., Ltd in full Shi Jin, and method is all carried out with reference to specification sheets.

One, the acquisition of corn WRKY family gene ZmWRKY33

Retrieval MAIZEGENOME and ncbi database, the supposition encoding sequence of acquisition ZmWRKY33, the primer of design amplification coding sequence, the upstream adds NcoI(CCATGG) restriction enzyme site, and the downstream adds Bst EII(GGTGACC) restriction enzyme site.Primer sequence is as follows:

Primer 1(upstream primer): 5 '-TGCTAACAATGAGGTATTAC-3 '

Primer 2 (downstream primer): 5 '-TTACCTCGGGTGGGGATTC-3 '

Extract the total RNA of tri-leaf period through the corn B73 self-mating system of arid processing, obtain cDNA by reverse transcription, the RT-PCR method, obtain full-length gene with top primer amplification, the gene size is 924bp, nucleotide sequence is shown in SEQ ID N0:2 in the sequence table, and coded proteinic aminoacid sequence is shown in SEQ ID N0:1 in the sequence table.

Concrete reaction is: get total RNA of 1 μ g (2 μ l), add 4 μ l Mgcl ₂(25mM), 2 μ l Reverse Transcription 10X Buffer, 2 μ l dNTP Mixture (10mM), 0.5 μ l Ribonuclease inhibitor (1u/ μ l), 1 μ l Oligo (dT) ₁₅Primer (0.5 μ g/ μ l), 1 μ l MMLV Reverse Transcriptase (15u/ μ l), 7.5 μ l Nuclease-Free Water, careful mixing, 42 ℃ of temperature are bathed 40min, then 95 ℃ of heating 5min, place 5min on ice, promptly obtain corresponding reverse transcription product cDNA.The cDNA that obtains is diluted 10 times, get the template of 2 μ l, under the guiding of primer 1 and primer 2, carry out pcr amplification as the PCR reaction.Reaction system is each 1 μ l of upstream and downstream primer (10mM), LA Taq enzyme (5u/ μ l) 0.5 μ l, dNTP(10mM) 1 μ l, 2 X GC bufferI, 25 μ l, ddH ₂O 19.5 μ l.The PCR reaction conditions is 94 ℃ of pre-sex change 5min, 94 ℃ of sex change 40s, and 55 ℃ of annealing 40s, 72 ℃ are extended 1min30s, 35 circulations, last 72 ℃ of 10 min.After reaction finishes, the PCR product is carried out 1% agarose gel electrophoresis to be detected, reclaim the also purpose fragment of purifying 945bp, it is cloned into the precious biotech firm of carrier pMD18-T Vector(), be transformed into again in the Trans5 α chemoreception attitude cell (full Shi Jin Bioisystech Co., Ltd), the PCR screening positive clone, check order, sequencing result shows that ZMWRKY33 has the dna sequence dna of SEQ ID N0:2 in the sequence table, SEQ ID N0:2 in the sequence table will contain the recombinant plasmid vector called after pMD18-ZmWRKY33 of ZmWRKY33 dna sequence dna by 924bp based composition.Sequencing result shows and has obtained the correct ZmWRKY33 gene of sequence.

Two, the Subcellular Localization of ZmWRKY33

With 5 '-CGGAGATCTATGAGGTATTACATTTATG-3 ' and 5 '-TTCTCTAGATTACCTCGGGTG

GGGATTC-3 ' is a primer, with pMD18-ZmWRKY33 plasmid is template, the complete encoding sequence of amplification ZmWRKY33, with the GFP downstream that is inserted into behind BglII, the XbaI double digestion on the PRTL2-mGFP that same enzyme cuts, the recombinant vectors called after PRTL2-ZmWRKY33-mGFP that sequence verification is correct.

PRTL2-ZmWRKY33-mGFP be used for onion ( Allium cepa) particle gun of epidermis transforms, compare with PRTL2-mGFP.Concrete steps are as described below:

1) preparation of plasmid: extract purifying PRTL2-ZMWRKY33-mGFP and PRTL2-mGFP(contrast with test kit) plasmid, concentration is 1mg/ml, is dissolved in H ₂O.

2) preparation of onion: onion is cut into the watermelon sheet, selects the comparatively smooth stem sheet of internal layer, strip the entocuticle cellular layer with tweezers, upset, light faces down, and is tiled in MS(100mg/L Amp) on the flat board, stand-by.

3) tungsten powder suspension preparation: take by weighing 60mg bronze (diameter 1.0um) in the 1.5mL centrifuge tube; Add the 1ml dehydrated alcohol, shook 1-2 minute, 10000rpm centrifugal several seconds; Abandon supernatant, repeat once; Suspend with the 1ml sterilized water, shook 1-2 minute, 10000rpm centrifugal several seconds; Abandon supernatant, repeat once; Suspend with the 1ml sterilized water, shake 1-2 minute, first with or be stored in-20 ℃ stand-by.

4) embedding of tungsten powder and DNA: get uniform tungsten powder suspension 50ul, add plasmid 12ul (1mg/ml), and mixing; Add 20ul 0.1M spermidine, mixing; Drop by drop add 50ul 2.5M CaCl lightly ₂,

And jiggle frequently; Behind the gentle concussion several minutes, room temperature was placed 10 minutes on the oscillator, and 10000rpm centrifugal 10 seconds, abandons supernatant, dehydrated alcohol rinsing 3 times; Again with particle again with particle suspension in the 30-15ul dehydrated alcohol, inhale with rifle and to beat mixing.

5) bombardment receptor material: get the 10ul uniform tungsten powder that suspends and place on the micro carrier.After treating that particle on the micro carrier is done, can load on the load plate of particle gun, notice that DNA faces down, (can split film 1100-1300Pa) so that launch on the MS flat board that is covered with onion epidermis.Utilize the PDS-1000 particle gun of Biorad company to enter onion epidermis with the tungsten powder bombardment of plasmid DNA.Bombardment operation:, adjust extremely suitably pressure (the high 200psi of more required at least pressure) (generally beat onion epidermis and select 1100psi for use) of the preceding black knob of tensimeter so steel cylinder pressure is selected about 1500psi with valve open on the steel cylinder.Place the super clean bench of instrument and open uv sterilisation in advance.Alcohol wipe chamber wall with 75% and interior object thereof.Micro carrier, can split film, woven wire and steep in advance in 75% alcohol and sterilize.With preceding, place clean aseptic plate to dry up.Assembling: with woven wire with can split in the knob that film is contained in the chamber top successively, put in the device of its lower floor and scribble tungsten powder and exsiccant macro carrier, DNA faces down.The plate of opening capping is placed on the pallet of lower floor.Close the upper wall door.Be evacuated to required vacuum tightness (being generally 26-28), bombardment.Treat that chamber recovers pressure, takes out sample and all parts.After finishing using, shut cylinder valve, vacuumize, bombard to steel cylinder upward pressure table and reduce to zero, get final product powered-down behind the answer chamber pressure.Black knob before the tensimeter is counterclockwise unscrewed.Plate is sealed, under white light or required light, cultivate after 30-40 hour for 22 ℃, under fluorescent microscope, observe, onion epidermis is cut into suitable size places the first water that drips on the slide glass, then cover glass is built, noted not having bubble. place then to observe under the fluorescent microscope and take pictures, determine that the external source fusion rotein is in intracellular position.

The result as shown in the figure, with fluorescent microscope under 445-490nm excitation light source irradiation, the cell that changes GFP empty carrier green-emitting fluorescence all in whole cell, and the cell that changes PRTL2-ZmWRKY33-mGFP fusion expression plasmid green-emitting fluorescence in nucleus only.The fusion rotein that shows ZmWRKY33 and GFP carrier is positioned in the nucleus, confirms that KT496 albumen has nuclear localization signal.A and B are PRTL2-mGFP carrier transient expression photo among the figure, and C and D are PRTL2-ZmWRKY33-mGFP carrier transient expression photo.A and C are the onion cell under the no excitation light irradiation, and B and D are the onion cell under the irradiation of 445-490nm excitation light source.

Three, the acquisition of ZmWRKY33 transgenic paddy rice

Will be with agrobacterium-mediated transformation by the gene relevant of embodiment one acquisition with resistance of reverse ZmWRKY33Rice transformation, concrete grammar is as follows:

1) transforms Agrobacterium

To scale off with NcoI and Bst EII enzyme with the ZmWRKY33 fragment that the RT-PCR amplification obtains and clones on the pMD18-T carrier, be connected carrier construction p-ZmWRKY33 with the carrier of cutting pCAMBIA1301 with same enzyme.Utilize the heat shock method to change above-mentioned recombinant vectors p-ZmWRKY33 over to Agrobacterium EH105 bacterial strain, utilize Agrobacterium that paddy rice is carried out cotransformation.

2) infect the rice callus tissue

Single colony inoculation of the positive reorganization Agrobacterium that the picking step 1) obtains is in the 20mL YEB liquid nutrient medium that contains kalamycin 50mg/L and Rifampin 50mg/L, under 28 ℃, 150rpm shaking culture 2-3 days, again in centrifugal 3 minutes of 4 ℃, 5000rpm, remove supernatant, bacterial sediment is resuspended in the AA substratum (Co (N0 that contains the 0.1mmol/L Syringylethanone ₃) ₂6H ₂O 0.15mg/L, CaCl ₂110mg/L, MgSO ₄122mg/L, KI 3.75mg/L, NaH ₂PO ₄H ₂O 150mg/L, Na ₂-EDTA 0.01mM, FeSO ₄7H ₂O 139mg/L, KCl 2.95g/L, MnSO ₄4H ₂O 84.5mg/L, ZnSO ₄7H ₂O 6.25mg/L, H ₃BO ₃5mg/L, Gly 37.5mg/L, CuSO ₄0.005mg/L, Na ₂MoO ₄2H ₂O 1.25mg/L, vitamin VB ₁5mg/L, nicotinic acid 5mg/L, pyridoxine hydrochloride VB ₆5mg/L, creatine 0.1g/L, casein hydrolysate 0.3g/L, L-Gln 87.6mg/L, L-Asp 26.6mg/L, sucrose 20g/L) in, 28 ℃ of following lucifuge shaking culture 1-2 hour to OD ₆₀₀=0.6-0.9.After good, the particulate state callus of fine growth conditions in paddy rice evening of selecting that method for plant tissue culture routinely obtains immerses and shakes 20 minutes in the reorganization Agrobacterium nutrient solution that above-mentioned conversion has p-ZmWRKY33, left standstill 30 minutes, take out, blot unnecessary bacterium liquid with aseptic filter paper, callus is inoculated in N ₆Be total to substratum (N ₆Substratum+10g/L glucose+1mg/L Syringylethanone+2,4 dichlorophenoxyacetic acid (2,4-D) 2mg/L) is gone up and is cultivated, wherein N ₆The prescription of minimum medium is (NH ₄) ₂SO ₄0.46g/L, KNO ₃2.83g/L, CaCl ₂0.2g/L, MgSO ₄0.092g/L, KH ₂PO ₄0.4g/L, Na ₂-EDTA 0.15g/L, FeSO ₄7H ₂O 0.11g/L, MnSO ₄4H ₂O 0.44g/L, ZnSO ₄7H ₂O 0.17g/L, H ₃BO ₃0.14g/L, CoCl ₂6H ₂O 0.0005g/L, CuSO ₄5H ₂O 0.0005g/L, Na ₂MoO ₄2H ₂O 0.005g/L, vitamin VB ₁0.01g/L, nicotinic acid 0.001g/L, pyridoxine hydrochloride VB ₆0.001g/L, creatine 0.1g/L, casein hydrolysate 0.3g/L, L-Pro 0.5g/L, sucrose 30g/L, agar 10g/L, pH5.8.After 2-3 days, callus is put into wide-necked bottle, use aseptic water washing 3-5 time, shake for several times at every turn, in the sterilized water that contains the 500mg/L cephamycin, soaked 30-60 minute then, use aseptic water washing 1 time at last again, place on the aseptic filter paper and dry, change screening culture medium (N at last over to ₆Minimum medium+2,4-D 2mg/L+ cephamycin 250mg/L) screening.

3) acquisition of positive transgenic rice plant

Rice callus after will infecting is organized in N ₆After cultivating 3-5 days altogether in the substratum altogether, go to the N that contains 50mg/L Totomycin and 400mg/L cephamycin ₆Solid medium (N ₆Macroelement+B ₅Trace element+B ₅Organic composition+300mg/L caseinhydrolysate+500mg/L proline(Pro)+30g/L sucrose+7-8g/L agar, pH 5.8) go up 3 weeks of screening, change the N that contains 50mg/L Totomycin and 200mg/L cephamycin again over to ₆4 weeks of screening change resistant calli over to pre-differentiation substratum (N subsequently on the solid medium ₆Substratum+1mg/L naa+2mg/L 6-benzylaminopurine+5mg/L dormin), in 3 weeks of illumination cultivation, change division culture medium (N again over to ₆Substratum+1mg/L naa+2mg/L 6-benzylaminopurine) breaks up on, move in the greenhouse regeneration plant that grows carried out root culture on strong seedling culture base (1/2 MS inorganic salt+0.5mg/L naa+0.25mg/L paclobutrazol) after, at 28 ℃, 15 hours/day illumination is cultivated after 1 month down and is got plant leaf, extract total DNA with the CTAB method, pcr amplification hygromix phosphotransferase (Hyg) gene under the guiding of forward primer 5 '-ACTCACCGCGACGTCTGT-3 ' and reverse primer 5 '-TTTCTTTGCCCTCGGACG-3 ' is through the positive transfer-gen plant of amplification acquisition 1009 bp dna fragmentations.Detected result shows with aforesaid method and has obtained to transform the transgenic paddy rice that p-ZmWRKY33 is arranged.

Four, ZmWRKY33 transgenosis T1 identifies for the plant drought tolerance

Results ZmWRKY33 T1 is for transgenic paddy rice seed, and choosing 6 above strains is to carry out drought stress.Its sprouting of 3 angels is cultivated in the water seed soaking, carries out resistance screening with the 50mg/L Totomycin then, simultaneously to change paddy rice fine the comparing in evening of empty carrier.Screen after 5 days, adjoining tree is all dead, and statistics transfer-gen plant resistance seedling and dead seedling number are analyzed the resistance of transfer-gen plant and separated ratio, selects resistance seedling and separating than the strain system that is about 3: 1 of non-resistant seedling to carry out the drought sieve.When treating seedling length, seedling is transferred to from culture dish in the triangular flask, when being cultured to tri-leaf period (the 3rd leaf unfolded fully), being carried out acute arid and handle to the 8cm left and right sides.Before the non-irrigated sieve shoot root portion is cleaned, select the consistent seedling of growth conditions (thickness, plant height), with thieving paper root water is blotted, put into the new triangular flask of exsiccant, every bottle 10 strain, every strain are screening 30 young plants.Every bottle of detail record arid time of origin, non-irrigated sieve process is 12 hours (decide on phenotype), rehydration then, the nutritive medium in the triangular flask was changed once in 2 days, with the fresh state of maintenance nutritive medium.The survival rate that contrasts late round-grained rice only is about 15%, and changes ZmThe survival rate of the strain system of WRKY33 is all more than 75%.

Five, ZmWRKY33 transgenosis T1 identifies for the plant salt tolerance

Step is the same before the screening.Carry out salt sieve with the Hoagland solution that contains 200mM NaCl, about 2 days, during change salts solution every other day one time.Handle after 2 days, contrast late fine plant and blade tip jaundice whiting occurs, the sagging or states such as half volume even full volume of blade, the flush away salts solution recovers normally to cultivate.Rehydration was changed one time of nutrition liquid in second day again, to remove remaining salinity.Nutritive medium in the triangular flask was changed once in 2 days, to keep the fresh state of nutritive medium. ZmWRKY33 transgenic paddy rice T ₁Can continued growth after generation, positive transgenic line recovered to cultivate, survival rate reaches more than 75%, and the yellow leaf of late fine japonica rice contrast (CK) plant, curl, withered, cane can not be upright, very fast death after recovering to cultivate, survival rate is less than 20%.

Six, ZmWRKY33 transgenosis T1 identifies for the plant resistance to cold

Step is the same before the screening.3 ℃ of cold sieves were handled 5 days, behind the full volume of contrast young leaves, Lao Ye, normally cultivate again, during nutritive medium in the triangular flask changed once in 2 days, to keep the fresh state of nutritive medium.The survival rate of transgenic line is all compared according to wanting high, and transgenic line can be grown in very fast recovery, and survival rate is all more than 60%, and the survival rate of adjoining tree is about 25%.

In a word, the T1 of the mistake express transgenic strain of corn WRKY33 transcription factor gene system shows that for the experiment of plant resistance of reverse the transgenosis survival rate more than 60%, illustrates corn substantially under adverse circumstance WRKY33Gene can significantly improve the resistance of transgenic line to low temperature, high salt and drought stress.

Claims (5)

1. corn WRKYGene is in the application that improves on the plant stress tolerance energy, and is described WRKYThe genes encoding following proteins: aminoacid sequence is SEQ ID N0:1 in the sequence table.

2. application according to claim 1 is characterized in that: with described WRKYGene transfered plant cell, tissue or organ will be cultivated into plant by plant transformed cell, tissue or organ again, obtain the transfer-gen plant that resistance of reverse improves.

3. application according to claim 2 is characterized in that: described WRKYGene is one of following nucleotide sequence: SEQ ID N0:2 or 3 dna sequence dna in the sequence table; Perhaps for having 90% above homology and the proteic dna sequence dna of coding identical function with SEQ ID N0:2 or 3 sequences.

4. application according to claim 3 is characterized in that; Described WRKYGene imports vegetable cell, tissue or organ by plant expression vector.

5. application according to claim 1 is characterized in that: described plant is that dicotyledons or unifacial leaf are planted

Thing.

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