CN103305485B - Plant stress tolerance related protein W106 and coding gene as well as application thereof - Google Patents

Abstract

The invention discloses a plant stress tolerance related protein W106 and a coding gene as well as application thereof. The protein provided by the invention is the following (a) or (b), wherein (a) is a protein formed by amino acid sequences shown in a sequence 1 in a sequence table; and (b) is a protein which is obtained by carrying out substitution and/or deletion and/or addition of one or more amino acid residues on the amino acid sequences in the sequence 1, has the same function and is derived from the sequence 1. Experiments prove that the gene W106 is expressed under induction of high salt, low temperature, ABS (abscisic acid) and drought. W106 can obviously improve the resistance of arabidopsis thaliana to NaCl and promote the development of root systems. The protein and gene provided by the invention provide a foundation for expression of genes related to artificial control of stress resistance and stress tolerance and can play an important role in culturing plants with enhanced stress resistance and stress tolerance.

Description

Plant stress tolerance correlative protein W106 and encoding gene thereof and application

Technical field

The present invention relates to biological technical field, particularly relate to a kind of plant stress tolerance correlative protein W106 and encoding gene thereof and application.

Background technology

The environment stresses such as arid, high salt and low temperature are the obstruction factors affecting wheat growth, growth.Therefore, understand wheat to the response of adverse environmental factor and signal transduction mechanism, improve the resistance of wheat breed, become one of vital task of wheat genetic research and wheat breed improvement.

A series of responsing reaction can be produced in plant materials, along with many Physiology and biochemistries and change developmentally under environment stress.Specify the reaction mechanism of plant to adverse circumstance, science argument will be provided for adversity gene engineering research and application.At present, plant stress-resistance Journal of Sex Research is deep into cell, molecular level gradually, and combines with genetics and genetic engineering research, and exploration biotechnology improves plant growth characteristics, its objective is and improves plant to the adaptive faculty of adverse circumstance.

Under the adverse environmental factor of the environment-stress such as arid, high salt and low temperature, a large amount of active oxygens (the reactive oxygen species produced in plant materials, ROS), ROS makes cell membrane lipid peroxidation with its extremely strong oxidisability, damage membranous system and oxidative cell, make lipid and protein metabolism extremely, finally growing of crop is caused serious injury.In long-term evolution process, the ROS purge mechanism that plant defines a set of enzymatic and non-enzymatic carrys out block film lipid peroxidation, reduces the injury that causes of environment to the full extent and existence.Selenoperoxidase (glutathioneperoxidases, GPX) is the crucial enzyme removing ROS in enzymatic purge mechanism.

Up to the present, GPXs gene has been cloned in the plants such as Arabidopis thaliana, corn, paddy rice, wheat, oat, rape, Radix Dauci Sativae, mung bean, spinach, Chinese cabbage, research shows, in plant materials, GPXs take part in and grows and stress response, and GPXs is subject to the abduction delivering of high salt, low temperature, physical abuse, heavy metal, ABA and oxidative stress on mRNA and protein level.Homology analysis shows, the GPXs in plant materials and the human phospholipid hydroperoxide glutathione peroxidase in animal body (phospholipid hydroperoxide GPxs, PHGPX) very high homology, all GPXs have the structural domain of high conservative.What is interesting is, plant GPXs coded by said gene aminoacid sequence in, seleno-cysteine (selenoCys) replace by halfcystine (Cys), and this selenoCys is considered to the catalytic site of animal PHGPX, this just greatly reduces the catalytic capability of GPXs in plant materials.In animal body the function of PHGPX mainly by catalyzing hydrogen peroxide and lipid peroxide also original Cell protection film from oxidative damage.Plant GPX family is very different with animal in structure, function and tissue distribution, and nearest research shows, plant GPXs gene family can reduce H equally ₂o ₂, organic hydroperoxide, phospholipid hydroperoxide, and higher catalytic activity is shown to the latter, can predict that GPXs can prevent membrane lipid peroxidation equally.Research finds, GPXs plays vital effect in the signal path resisting environment stress.The isozyme of 8 kinds of GPXs is had been found that in Arabidopis thaliana, molecular weight of albumen is at about 20kD, be distributed on 4 karyomit(e)s, be positioned in the organoids such as tenuigenin, chloroplast(id), plastosome, in abiotic stress, different GPXs gene regulated by unlike signal transduction pathway, but concrete molecular mechanism is not clear.Research shows, the abiotic stress such as high salt, heavy metal, low temperature, arid, physical abuse all can induce the expression of GPXs, GPXs may participate in the crisscross network of response the external environment cell signaling, cell aging etc. of coercing, and improves the adaptive faculty of plant adverse circumstance to external world.

Stress tolerance due to plant is the complex network regulated and controled by polygene, and the ROS how plant experiences, utilizes and remove the generation of abiotic stress process has important theory significance and practical value.Rely on and import gene crucial in environment stress, promote the expression of multiple genes involved, activate whole signal network, thus strengthen the resistance of plant, become the engineered study hotspot of plant stress-resistance.

Summary of the invention

An object of the present invention is to provide a kind of plant stress tolerance correlative protein W106 and encoding gene thereof.

A kind of albumen provided by the invention is Selenoperoxidase, derives from Triticum wheat (Triticumaestivum L.), is following (a) or (b):

A protein that () is made up of the aminoacid sequence shown in sequence in sequence table 1;

B the aminoacid sequence of sequence 1 is had the protein derived by sequence 1 of identical function through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation by ().

In above-mentioned albumen, the replacement of one or several amino-acid residue and/or disappearance and/or be added to the replacement and/or disappearance and/or interpolation that are no more than 10 amino-acid residues.

In above-mentioned sequence table, the amino acid residue sequence of sequence 1 is made up of 171 amino-acid residues.

The gene of above-mentioned albumen of encoding also is the scope of protection of the invention.

Said gene is following 1)-6) in any DNA molecular:

1) DNA molecular shown in sequence 2 in sequence table;

2) in sequence table, sequence 2 holds the DNA molecular shown in the 160 to 675 Nucleotide from 5 ';

3) in sequence table, sequence 2 holds the DNA molecular shown in the 132 to 675 Nucleotide from 5 ';

4) in sequence table, sequence 2 holds the DNA molecular shown in the 157 to 672 Nucleotide from 5 ';

5) under strict conditions with 1) or 2) or 3) DNA sequence dna that limits hybridizes and encode and the DNA molecular of plant stress tolerance correlative protein;

6) with 1) or 2) or 3) DNA sequence dna that limits has more than 90% homology, and the DNA molecular of coding and plant stress tolerance correlative protein.

Above-mentioned stringent condition is in the solution of 6 × SSC, 0.5%SDS, and hybridize at 65 DEG C, then use 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.

Above-mentioned sequence 2 is made up of 839 Nucleotide, and open reading frame is from 5 ' end the 160 to 675 bit base.

Recombinant expression vector containing said gene, expression cassette, transgenic cell line or recombinant bacterium are also the scope of protection of the invention.

Above-mentioned recombinant expression vector is said gene is inserted the recombinant vectors obtained in pBI121, SmaI and the SpeI enzyme be specially the sequence 2 of sequence table inserts pBI121 from the DNA fragmentation shown in 5 ' end the 132 to 675 Nucleotide cuts the recombinant plasmid obtained between recognition site.

The amplification total length of said gene or the primer pair of its any fragment are also the scope of protection of the invention, and above-mentioned primer pair is made up of the DNA molecular shown in sequence 4 in the DNA molecular shown in sequence in sequence table 3 and sequence table.

Above-mentioned albumen or said gene or the application of above-mentioned recombinant expression vector in regulating plant resistance of reverse and/or resistance of oxidation are also the scope of protection of the invention.

In above-mentioned application, above-mentioned resistance to against being specially salt tolerant or drought-resistant; Above-mentioned regulation and control are specially raising.

Above-mentioned albumen is also the scope of protection of the invention as the application in Selenoperoxidase.

Another object of the present invention is to provide a kind of method of cultivating transgenic plant.

Method provided by the invention said gene is imported in object plant,

Obtain transgenic plant; Described transgenic plant have following 1) or 2) feature:

1) described transgenic plant resistance of reverse is higher than described object plant;

2) described transgenic plant resistance of oxidation is higher than described object plant.

Said gene is imported in described object plant by above-mentioned recombinant expression vector.

In aforesaid method, described resistance to inverse be salt tolerant or drought-resistant;

In aforesaid method, above-mentioned purpose plant is dicotyledons or monocotyledons, and in an embodiment of the present invention, described dicotyledons is specially Arabidopis thaliana.

Described salt tolerant is embodied as under salt stress, and the root of described transgenic plant is grown up in described object plant;

Describedly anti-oxidantly to be embodied as at H ₂o ₂coerce down, the growth that is more flourishing than described object plant and/or described transgenic plant of the root system of described transgenic plant does not suppress by obvious;

The described drought-resistant ABA that is embodied in coerces down, and the germination rate of described transgenic plant is higher than described object plant.

Experiment of the present invention proves, the gene W106 that the present invention finds is at high salt, low temperature, ABA, H ₂o ₂express with under the induction of arid, by transgenic technology, obtain turning W106 plant, prove that W106 can significantly improve Arabidopis thaliana to NaCl, ABA and H ₂o ₂resistance, promote the growth of root system.Albumen provided by the invention and gene are that manual control expression that the is degeneration-resistant and gene of resistance to retrocorrelation provides the foundation, and play an important role in the plant cultivating resistance and resistance of reverse enhancing.

Accompanying drawing explanation

Fig. 1 is the expression pattern of W106 gene under various coercing

Fig. 2 is wild-type and transgenic arabidopsis growing state under NaCl process

Fig. 3 is wild-type and transgenic arabidopsis growing state under exogenous aba treatment

Fig. 4 is external source H ₂o ₂the lower wild-type of process and transgenic arabidopsis growing state

Fig. 5 is SDS-PAGE electrophoresis detection PET-W106 object band

Fig. 6 is the GPX enzyme assay of W106 albumen

Embodiment

The experimental technique used in following embodiment if no special instructions, is ordinary method.

Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.

% in following embodiment, if no special instructions, is mass percentage.

The clone of embodiment 1, W106

One, the clone of W106

By the hydroponics growing common wheat of about 10 days (Triticum aestivum L.) kind little Bai wheat (Triticumaestivum cv.Xiaobaimai, the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science, and the public also can obtain (being numbered ZM242) from national germplasm resource bank, mention that the document of little Bai wheat is: the screening of Sun Haitao etc., wheat TaDREB6 transcription factor interact protein, Scientia Agricultura Sinica, 2011,44 (22): 4740-4747., mention that the document of little Bai wheat is: Isolation and molecular characterization of the Triticum aestivumL.ethylene-responsive factor 1 (TaERF1) that increases multiple stresstolerance, Plant Mol Biol (2007) 65:719-732, Zhao Shi Xu, Lan Qin Xia, MingChen, Xian Guo Cheng, Rui Yue Zhang, Lian Cheng Li, Yun Xing Zhao, Yan Lu, Zhi YongNi, Li Liu, Zhi Gang Qiu, You Zhi Ma) seedling NaCl process in tri-leaf period 2 hours, with liquid nitrogen flash freezer,-80 DEG C save backup.

Adopt Trizol method (TianGen) to extract wheat leaf blade total serum IgE, the first chain cDNA synthesizes with ThermoScript II XL (AMV).Adopt SMART method synthesis ds cDNA, PCR primer carries out 1.0% agarose gel electrophoresis detection.

The sequence 2 in sequence table is obtained by the method for 5 ' RACE and 3 ' RACE.

In this sequence table, the unnamed gene shown in sequence 2 is W106 gene, its open reading frame is 5 ' end the 160th-675 Nucleotide of the sequence 2 from sequence table, be W106 albumen by the protein designations of this genes encoding, be made up of 171 amino-acid residues, its aminoacid sequence is the sequence 1 in sequence table.

Above-mentioned sequence 2 also can pass through synthetic.

Two, real-time fluorescence quantitative PCR analyzes the expression characterization of W106 after Stress treatment

Seedling age is the little Bai wheat seedling of 10 days, carries out following process:

(1) Osmotic treatment: the wheat seedling of water planting is taken out the moisture blotted on root, be placed on dry filter paper, arid is cultivated after 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours and is taken out material, and with liquid nitrogen flash freezer ,-80 DEG C save backup.

(2) salt marsh process: wheat seedling is placed in 2% by NaCl and Na ₂sO ₄(NaCl and Na in the sodium salt solution of composition ₂sO ₄mass percent be 3: 2) in, illumination cultivation takes out material after 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours respectively, and with liquid nitrogen flash freezer ,-80 DEG C save backup.

(3) dormin process: dormin (ABA) solution wheat seedling being placed in 200 μMs, illumination cultivation is taken out respectively after 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours and is used liquid nitrogen flash freezer, and-80 DEG C save backup.

(4) H ₂o ₂process: H wheat seedling being placed in 100mM ₂o ₂in solution, illumination cultivation is taken out respectively after 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours and is used liquid nitrogen flash freezer, and-80 DEG C save backup.

(5) subzero treatment: at wheat seedling being placed in 42 DEG C, illumination cultivation is taken out respectively after 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours and is used liquid nitrogen flash freezer, and-80 DEG C save backup.

(6) process contrasted: directly get without the wheat seedling-80 DEG C of any process frozen in contrast (0 hour).

Adopt Quikprep Micro mRNA Purification Kit (Pharmacia) to carry out the separation of mRNA the seedling seedling of above-mentioned each process, then adopt R103-Quant_Reverse_Transcriptase (TIANGEN) to be cDNA by the mRNA reverse transcription of purifying.

According to W106 sequence, in its variable region design special primer W106RTF and W106RTR (product size is 573bp).Take actin as reference gene, primer is actin-2F and actin-2R.

W106RTF：5’-GGAAACATTACAGTATTGCTGAGAGCAAC-3’；

W106RTR：5’-CGCATGATCTTCCCTGGAGATATA-3’。

actin-2F：5’-CTCCCTCACAACAACCGC-3’；

actin-2R：5’-TACCAGGAACTTCCATACCAAC-3’。

With the above-mentioned cDNA managed for template, carry out real-time fluorescence quantitative PCR with W106RTF and W106RTR everywhere.

As shown in Figure 1, A is the result of salt marsh process to result, and B is the result of ABA process, and C is the result of Osmotic treatment, and D is the result of subzero treatment, and E is H ₂o ₂the result of process, can find out, plant is subject to salt marsh, ABA, arid and H ₂o ₂in the early response stage of process, W106mRNA just starts to transcribe in a large number; Under subzero treatment, W106mRNA expression amount raises to some extent again after 1h falls after rise.

Embodiment 2, W106 are on the impact of plant stress tolerance

One, the structure of recombinant expression vector

1, the clone of W106 gene

According to the primers of TaTPRPK1 gene to (W106-121F and W106-121R), prime end is introduced SmaI and SpeI enzyme respectively and is cut recognition site.

W106-121F:5 '-CCCGGGAAACATTACAGTATTGCTGAGAGC-3 ' (sequence 3);

W106-121R:5 '-ACTAGTCAAACCTCCAACAGCTTCTTG-3 (sequence 4).

Extract little Bai wheat blade total serum IgE, it is template that reverse transcription obtains cDNA, increases with above-mentioned primer, and the PCR primer obtained carries out 1.2% agarose gel electrophoresis, and its size is 0.5Kb.This PCR primer is sent to order-checking, there is sequence 2 in sequence table and hold 132-675 position Nucleotide from 5 ', be W106 gene.

Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa company, Code No.:DV807A) is adopted to reclaim the PCR primer of purifying 0.5Kb size.

2, the structure of recombinant expression vector

1. use Restriction enzyme Sma I and SpeI (Promega, R6121, R6591) enzyme to cut the PCR primer that step 1 reclaims purifying, reclaim digestion products;

2. use Restriction enzyme Sma I and SpeI cleaving plant carrier for expression of eukaryon pBI 121 (purchased from Clontech company), reclaim carrier framework;

3. step digestion products is 1. connected with step carrier framework 2.;

4. by the electroporated TOP10 bacterial strain (Tiangen, CB104-03) of step connection product 3., 37 DEG C of incubated overnight, picking positive colony extracts plasmid and checks order.

Sequencing result shows, this plasmid is for holding the DNA fragmentation shown in the Nucleotide of 132-675 position to insert the carrier obtained between SmaI and the SpeI restriction enzyme site of pBI121 carrier, by this plasmid called after pBI121-W106 from 5 ' sequence 2 in sequence table.

Two, the acquisition of transgenic plant

1, with above-mentioned recombinant plasmid pBI121-W106 transformation Agrobacterium C58 (purchased from Beijing Baeyer enlightening biotech company), recombinational agrobacterium is obtained.

The plasmid extracting recombinational agrobacterium sends to order-checking, and this plasmid of result is pBI121-W106, proves that this recombinant bacterium is positive recombinational agrobacterium, by its called after C58/pBI121-W106.

2, recombinational agrobacterium C58/pBI121-W106 is inoculated in YEP liquid nutrient medium, 28 DEG C, 3000rpm cultivate about 30 hours;

3, the bacterium liquid of step 2 is gone in YEP liquid nutrient medium (containing 50 μ g/ml Rifampins), 28 DEG C, 300rpm cultivates about 14 hours (bacterium liquid OD600 reaches 1.5-3.0);

4, collect thalline, 4 DEG C, the centrifugal 10min of 4000g, be diluted to OD600 with 10% sucrose (containing 0.02%silwet) and be about 1.0;

5, by Arabidopis thaliana Columbia ecotype Col-0 (U.S.'s Arabidopis thaliana information resource network, hereinafter referred to as wildtype Arabidopsis thaliana, purchased from SALK company) whole strain tips upside down in the container of the bacterium liquid filling step 4 together with flowerpot, makes flower soak about 50s, after immersion, take out flowerpot, be sidelong in pallet, cover black plastic cloth, plastic cloth is opened after 24hr, upright placing flowerpot, carries out normal illumination cultivation, mixed receipts T ₀in generation, turns W106 Arabidopis thaliana seed.By T ₀4 strain T are obtained for turning after on W106 Arabidopis thaliana planting seed to the MS substratum containing 50mg/ml kantlex ₁in generation, turns W106 Arabidopsis thaliana Seedlings.

Adopting uses the same method proceeds to wildtype Arabidopsis thaliana by empty carrier pBI121, obtains T ₁in generation, turns empty carrier Arabidopis thaliana.

Respectively by T ₁in generation, turns W106 Arabidopis thaliana and T ₁in generation, turns the sowing of empty carrier Arabidopis thaliana, selfing, until obtain T ₃in generation, turns W106 Arabidopis thaliana and T ₃in generation, turns empty carrier Arabidopis thaliana.

Extract T ₃in generation, turns the DNA of W106 Arabidopsis plant, and based on carrier pBI121 sequence, design primer (121F:5 '-GACGCACAATCCCACTAT CC and 121R:5 '-AATCATCGCAAGACCGGC) carry out PCR detection, that obtain 500bp is positive T ₃in generation, turns W106 Arabidopis thaliana.

Extract T ₃for the genomic dna turning empty carrier Arabidopis thaliana, be that primer carries out pcr amplification with W106-121F and W106-121R, do not obtain the object fragment of 500bp, T is described ₃in generation, turns empty carrier Arabidopis thaliana and successfully constructs.

Three, the resistance of reverse qualification of transgenic plant

Respectively by T ₃in generation, turns W106 Arabidopis thaliana (W106) seed, T ₃in generation, turns empty carrier Arabidopis thaliana seed and wildtype Arabidopsis thaliana seed (WT) is planted on common MS substratum, grows after 7 days, carries out following resistance of reverse qualification, the strain of each strain 10, tests in triplicate, results averaged:

1, Ficus caricaL (salt tolerant experiment): the MS substratum (NaCl concentration is respectively 0mM, 50mM, 100mM, 150mM, 200mM) transferred to by the growth seedling of 7 days being in identical vegetative period containing NaCl above continues cultivation 7 days.

Observe growing state, measure that to grow the Arabidopsis thaliana Seedlings root of 7 days at the MS substratum containing NaCl long, as shown in Figure 2,2a is the growing state of WT and transgenic arabidopsis under 150mM NaCl process to result; 2b be under the process of different N aCl concentration WT and transgenic arabidopsis root long; Find out from Fig. 2 a, under the MS substratum (normal growth) of 0mMNaCl, T ₃the root that generation turns W106 Arabidopis thaliana (W106) and wildtype Arabidopsis thaliana is long similar, and under the MS substratum of 150mMNaCl, T ₃for long about 2 times of the long comparatively WT strain of the root turning W106 Arabidopis thaliana (W106).

Concrete length is shown in Fig. 2 b,

T ₃generation turn the root of W106 Arabidopis thaliana (W106) in the MS substratum containing 0mM, 50mM, 100mM, 150mM, 200mM NaCl grow be respectively 5.01,3.80,4.00,2.83,0.85cm;

The root of wildtype Arabidopsis thaliana in the MS substratum containing 0mM, 50mM, 100mM, 150mM, 200mM NaCl be long is respectively 5.03,4.21,2.73,1.16,0.77cm;

T ₃in generation, turns the result of empty carrier Arabidopis thaliana and wildtype Arabidopsis thaliana without significant difference.

T is described ₃in generation, turns W106 Arabidopis thaliana (W106) salt tolerant.

2, ABA process (drought-resistant experiment): cultivate on planting seed to the MS substratum of 0.8 μM of ABA 14 days, not add ABA for contrast, observes growing state and calculates germination rate.

As shown in Figure 3, a is the growing state of WT and transgenic arabidopsis under 0.8 μM of ABA process to result; B is that under 0.8 μM of ABA process, WT and transgenic arabidopsis germination rate are added up, and finds out from 3a, and left figure does not add T under ABA ₃in generation, turns the growth of W106 Arabidopis thaliana (W106) and wildtype Arabidopsis thaliana without significant difference, and right figure is after 0.8 μM of ABA process, and the growth of wild-type plant is obviously suppressed, and starts yellow.

Concrete data are shown in shown in Fig. 3 b, and ABA concentration is 0.8 μM time, and wild type seeds germination rate is only 10%, and T ₃it is 38% that generation turns W106 Arabidopis thaliana (W106) germination rate, and T is described ₃it is drought-resistant that in generation, turns W106 Arabidopis thaliana (W106).

T ₃in generation, turns the result of empty carrier Arabidopis thaliana and wildtype Arabidopsis thaliana without significant difference.

3, H ₂o ₂process (resistance of oxidation experiment): the growth seedling of 7 days being in identical vegetative period is transferred to containing 1mM H ₂o ₂mS substratum on continue cultivation 7 days, observe growing state.

Result as shown in Figure 4, can be found out, is 1mM H through concentration ₂o ₂process, T ₃in generation, turns W106 Arabidopis thaliana (W106) plant and grows vigorous than WT lines (WT), and define more flourishing root system system, grow not suppressed, and wildtype Arabidopsis thaliana blade starts to turn yellow, root system is also undeveloped, illustrates that the resistance of oxidation of transfer-gen plant is higher than WT lines.

T ₃in generation, turns the result of empty carrier Arabidopis thaliana and wildtype Arabidopsis thaliana without significant difference.

The enzyme assay of embodiment 3, W106

1, the preparation of W106 albumen

According to W106 sequence, design special primer W106-PETF and W106-PETR, prime end is introduced BamHI and XhoI enzyme respectively and is cut recognition site.

W106-PETF 5’-TACGGATCCAACATGGGTGCGGCAGAGT-3’

W106-PETR 5’-TAGCTCGAGAACCTCCAACAGCTTCTTGATG-3’

With sequence in sequence table 2 for template, be that primer carries out pcr amplification with W106-PETF and W106-PETR, obtain the PCR primer W106 of 0.5kb.

PCR primer is cut with BamHI and XhoI enzyme, connect with prokaryotic expression carrier pET-28a (+) (Novagen, the 69864-3) that cut through same enzyme, obtain connecting product, proceed to E.coli BL21 (DE3) fungus strain, obtain transformant.

Extract the plasmid of transformant, send to order-checking, this plasmid is for holding the DNA fragmentation shown in the Nucleotide of 157-672 position to insert the carrier obtained between BamHI and the XhoI restriction enzyme site of PET-28a carrier from 5 ' sequence in sequence table 2, by this plasmid called after PET-28a-W106, by recombinant bacterium called after BL21 (the DE3)/PET-28a-W106 containing this plasmid.

By above-mentioned BL21 (DE3)/PET-28a-W106 in 40ml LB (containing 50mg/ml kanamycin) substratum 37 DEG C be cultured to D ₆₀₀=0.5, add 108mM IPTG, transfer to 22 DEG C of inductions 4 hours respectively, 3000g collects thalline in centrifugal 10 minutes, resuspended in 100mM Tris/HCl (pH 8) solution, sonicated cells wall, 4 DEG C, 15000g collects supernatant liquor (W106) in centrifugal 15 minutes.

Adopting uses the same method proceeds to BL21 (DE3) by empty carrier PET-28a, obtains BL21 (DE3)/PET-28a, induces latter 4 hours, obtain empty carrier supernatant liquor (PET-28a).

Supernatant liquor (W106) and empty carrier supernatant liquor (PET-28a) are carried out purifying respectively, method is with reference to His label protein purification kit (Cowin, CW0009), purifying W106 (the soluble proteins W106 with histidine-tagged) and reference protein (PET-28a) (histidine-tagged protein of expression) is obtained respectively.

Albumen after supernatant liquor before above-mentioned purifying and purifying is carried out SDS-PAGE electrophoresis detection.

As shown in Figure 5, supernatant liquor (W106) and purifying W106 all obtain the target protein that size is 19kD to result.

2, the enzyme of W106 albumen is lived and is identified

Under reductive agent gsh exists, with H ₂o ₂for substrate, under 412nm optical wavelength, measure enzymic activity, method is with reference to Flohe and Gunzler (1984).Respectively get 0.1ml supernatant to note in enzyme pipe and non-enzymatic pipe, and the heating of non-enzymatic pipe is made enzyme deactivation, add 1mM reduced glutathion (Sigma, G4251) 0.2ml and the 1.5mM H through 37 DEG C of preheatings respectively ₂o ₂1.5ml, reacts 3m in immediately at 37 DEG C, then in 2 test tubes, add the metaphosphoric acid precipitated liquid 1.6ml (1.67%HPO of 1.67% ₃, 0.05%EDTA-2Na, 28%NaCl).The centrifugal 10min of 3000rpm, retain supernatant liquor, separately get 2 test tubes and add supernatant liquor (W106 respectively, protein concentration is 10.18mg/ml) or empty carrier supernatant liquor (PET-28a, protein concentration is 6.34mg/ml) 0.5ml, get 1 test tube again and add the metaphosphoric acid precipitated liquid 1.6m l of distilled water 0.4m l and 1.67% as blank tube, and in these 3 test tubes, respectively add the Na of 0.32M ₂hPO ₄1ml and 5, two (2-nitrobenzoic acid) (DTNB, Sigma, the D8130) 0.5ml of 5 '-two sulphur, reaction 5m in, reads OD value in 412nm place colorimetric.The reduction that in the vigor available units time of Selenoperoxidase, catalysis GSH is oxidized represents, unit of enzyme activity (U), 1U=μm of ol min ^-1(mg protein) ^-1.Glutathione peroxidase activity (U)=[(non-enzymatic pipe OD ₄₁₂-enzyme pipe OD ₄₁₂) × A × 6.25]/[in 5 (min) × 1ml sample liquids protein (mg)].

Result as shown in Figure 6, can be found out,

The enzyme work of empty carrier supernatant liquor (PET-28a) is 0, and the enzyme work of supernatant liquor (W106) (PET-W106,4h) is 0.04 μm of ol min ^-1(mg protein) ^-1; Illustrate that W106 is Selenoperoxidase (GPX).

Illustrate that W106 albumen has GPX activity in plant materials, participate in NaCl, H ₂o ₂coerce etc. the peroxidation caused.

Claims (5)

1. albumen W106 is improving the application in plant stress tolerance and/or resistance of oxidation; Described resistance to inverse be salt tolerant or drought-resistant; The aminoacid sequence of described albumen W106 is sequence 2 in sequence table.

2. albumen W106 encoding gene is improving the application in plant stress tolerance and/or resistance of oxidation; Described resistance to inverse be salt tolerant or drought-resistant; The nucleotides sequence of described albumen W106 encoding gene is classified as sequence 1 in sequence table.

3. the recombinant expression vector containing albumen W106 encoding gene is improving the application in plant stress tolerance and/or resistance of oxidation; Described resistance to inverse be salt tolerant or drought-resistant;

Described recombinant expression vector is that described albumen W106 encoding gene is inserted the recombinant vectors obtained in pBI121;

The aminoacid sequence of described albumen W106 is sequence 2 in sequence table.

4. cultivate a method for transgenic plant, be that albumen W106 encoding gene is imported in object plant, obtain transgenic plant; Described transgenic plant have following 1) or 2) feature:

1) described transgenic plant resistance of reverse is higher than described object plant;

2) described transgenic plant resistance of oxidation is higher than described object plant;

The aminoacid sequence of described albumen W106 is sequence 2 in sequence table;

Described resistance to inverse be salt tolerant or drought-resistant.

5. method as claimed in claim 4, is characterized in that:

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