CN102234318A

CN102234318A - Plant stress tolerance related protein TaTPRPK1, encoding gene thereof, and application thereof

Info

Publication number: CN102234318A
Application number: CN2010101616099A
Authority: CN
Inventors: 徐兆师; 马有志; 李连城; 陈明
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2010-04-27
Filing date: 2010-04-27
Publication date: 2011-11-09
Anticipated expiration: 2030-04-27
Also published as: CN102234318B

Abstract

The invention discloses a plant stress tolerance related protein TaTPRPK1, an encoding gene thereof, and an application thereof. The protein provided by the invention is (a) or (b): (a) the protein is composed of an amino acid sequence represented by a sequence 1 in a sequence listing; and (b) the protein which is related to the plant stress tolerance, derived from the sequence 1, and obtained through substituting and/or deleting and/or adding to the amino acid sequence of the sequence 1 by one or some amino acid residues. The invention also discloses the encoding gene (TaTPRPK1) of the protein, and the application of the protein in breeding stress tolerance plants. The TaTPRPK1 can enhance the resistance of plants to NaCl, and the growth of roots. The TaTPRPK1 of the present invention lays the foundation for artificially controlling the expression of the anti-stress and stress resistance related gene, and plays an important role in breeding plant varieties with strengthened anti-stress and stress resistance.

Description

Plant stress tolerance correlative protein TaTPRPK1 and encoding gene thereof and application

Technical field

The present invention relates to a kind of plant stress tolerance correlative protein TaTPRPK1 and encoding gene and application.

Background technology

Environment stresses such as arid, high salt and low temperature are the obstruction factors that influences wheat growth, growth.Therefore, the understanding wheat is replied and signal transduction mechanism adverse environmental factor, improves the resistance of wheat breed, becomes one of vital task of wheat genetic research and wheat breed improvement.

Under environment stress, can produce a series of responsing reactions in the plant materials, the variation that is accompanied by many Physiology and biochemistries and grows.Clear and definite plant is to the reaction mechanism of adverse circumstance, will provide the science argument for adversity gene engineering research and application.At present, the plant stress-resistance Journal of Sex Research is deep into cell, molecular level gradually, and combines with genetics and genetic engineering research, explores and improves plant growth characteristics with biotechnology, its objective is and improves the adaptive faculty of plant to adverse circumstance.

Under the adverse environmental factor of environment-stress such as arid, high salt and low temperature, plant can be made corresponding adjustment on molecule, cell and integral level, with the injury that reduces environment to the full extent and caused and survived.Many genes are expressed by stress-inducing, the product of these genes not only can be participated in the stress response of plant directly, and can regulate other Expression of Related Genes or participate in signal transduction path, thereby plant is avoided or reduce injury, strengthen coercing the resistance of environment.With coerce relevant gene product and can be divided into two big classes: the product of first kind genes encoding comprises that ionophorous protein, aquaporin, the osmoregulation factor (sucrose, proline(Pro) and trimethyl-glycine etc.) synthetic enzyme etc. participate in the gene product that plant stress is replied directly; The product of second genoid coding comprises the protein factor that participates in coercing relevant signal transmission and genetic expression adjusting, as protein kinase, transcription factor etc.Wherein, protein kinase plays an important role in the regulation and control of the perception of plant stress signal, transmission.

Up to the present, the protein kinase of having found has about 300 kinds approximately, the catalytic structure district that intramolecularly all exists a homologous to be made of about 270 amino-acid residues.In systems such as cell signaling, cell cycle regulating, protein kinase has formed crisscross network.This quasi-enzyme catalytic migrates out phosphoric acid and is covalently bound on the hydroxyl of some Serine, Threonine or tyrosine residues the specified protein molecule from ATP, thereby changes the conformation and the activity of protein, enzyme.

At present, in plant known with coerce relevant protein kinase and mainly contain: serine/threonine protein kitase such as MAPK (mitogen-activated protein kinase), CDPK (calcium-dependent proteinkinase); Tyrosine protein kinase such as Src family, Tec family, ZAP70, family, JAK family; Also has a kind of serine/threonine/tyrosine dual specificity protein kinases, the protein kinase relevant, CDC25 etc. as DYRK.The signal conductive process that this three proteinoids kinases all has the corresponding external environment of bibliographical information involved in plant to coerce improves the plant adaptive faculty of adverse circumstance to external world.

According to the kind of the phosphorylated amino acid residue of protein kinase, protein kinase can be divided into the Ser/Thr protein kinase, Tyr protein kinase and dual specific Ser/Thr/Tyr protein kinase.The Ser/Thr protein kinase mainly comprises MAPK, CDPK, JNK etc.It is more that such protein kinase is studied at present, and play an important role in signal transduction path.And tyrosine protein kinase is divided into two classes, and a class is the non-receptor type tyrosine protein kinase, is representative with the src gene product, also has Yes, Fyn, Lck, Fgr, Lyn, Fps/Fes and Ab1 etc. in addition; Another kind of is the receptor type tyrosine protein kinase, and according to their structure difference, receptor type tyrosine kinase can be divided into 9 types.In zooblast, tyrosine protein kinase often participates in important cells behaviors such as apoptosis, canceration.Tyrosine protein kinase research is later in the plant.People's such as Maya Mayrose work shows that the dual-specificity kinase LeMPK3 in the tomato can improve the opposing of plant to pathogenic agent.Document proposes, and after the infringement that is subjected to pathogenic agent, the kinase whose activity of LeMPK3 at first improves, the also of short duration raising of the expression amount of the mRNA of LeMPK3 then.2002, people such as ParvathiRudrabhatla obtained a dual specificity protein kinases from peanut cDNA library, have serine/threonine/protein tyrosine kinase activity.And find that this kinase whose mRNA expression level and kinase activity are subjected to inducing of low temperature and high-salt stress.But its protein expression level does not but have obvious variation.NaCl in 250mM concentration handles under the 24h, and the mRNA expression level of STY has improved 20 times, handles 24h under 4 ℃ of conditions, and its mRNA expression level has improved about 50 times.Under 100mMABA handled, the STY expression amount did not change.

At present, it is disease-resistant to find all that in many plants protein kinase participates in, signal transduction process such as degeneration-resistant border (ParvathiRudrabhatla, 2002; Lizhong Xiong, 2003).Parvathi Rudrabhatla etc. thinks that a kind of STY kinases in the peanut has participated in peanut the start-up phase of abiotic stress is answered, and has proved that a kind of protein kinase cascade signal transmittance process of non-MAPK approach plays an important role in abiotic stress.People such as Lizhong Xiong think that rice protein kinase OsMAPK5 can improve paddy rice to low temperature, arid, the resistance of high salt and disease etc.And Jen Sheen thinks, the protein kinase C DPK (CDPK1 and CDPK1a) that calcium ion relies on can activate the promotor with plant stress-resistance border genes involved, involved in plant adverse circumstance signal conductive process.Reports such as Riichiro Yoshida, ABA inductive protein kinase SnRK2 plays important effect in the signal conductive process of plant responding desiccation stress in Arabidopis thaliana.Because the stress tolerance of plant is the complex character by the polygene regulation and control, rely on to import the comprehensive raising that the individual feature protein gene is difficult to realize stress resistance of plant.Therefore, utilize a key transcription factor to promote the expression of a plurality of functional genes, thereby strengthen the resistance of plant, become the engineered research focus of plant stress-resistance.

Comprehensive present result of study, protein kinase occupies an important position in the signal transduction path of plant responding environment stress.Protein kinase improves or reduces self kinase activity by self phosphorylation, improves or reduce the enzymic activity of substrate protein by phosphorylated substrate, and the substrate protein enzyme of regulating and control downstream gene is again lived then, forms a cascade regulatory pathway.As RAS signals-modulating approach, experience the signal of part in the cell receptor tyrosine protein kinase after, form the concurrent body phosphorylation of being conigenous of dimer, activate RAS, activate the cascade reaction that causes protein kinase by activatory RAS then.This cascade regulation and control are a kind of very accurate very complicated regulated and control networks, and protein kinase may exercised its function as a kind of switch in the upstream.

Summary of the invention

The purpose of this invention is to provide a kind of plant stress tolerance correlative protein TaTPRPK1 and encoding gene and application.

Protein TaTPRPK1 provided by the invention is a kind of dual specificity protein kinases, derives from Triticum wheat (Triticum aestivum L.), is following (a) or (b):

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;

(b) with the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant stress tolerance by sequence 1 deutero-protein.

The protein that protein is made up of 494 amino-acid residues shown in the sequence 1; from aminoterminal the 1st to 7 amino acids residue is N end myristoylation site; the the 63rd to 312 amino acids residue is conservative serine/threonine/tyrosine protein kinase active region, is C end TPR conserved regions from aminoterminal the 392nd to 481 amino acids residue.The TaTPRPK1 kinases contains the total conservative territory of 11 protein kinases, and the kinases conserved regions is from the 68th amino acids to the 312 amino acids, and span is 245 amino acid.The Semen Myristicae site of N end has film grappling function, and albumen is positioned on the cytolemma inner membrance.The major function of the TPR conserved regions of C end is to assist protein-interacting, also has pilot protein to be positioned at function on the nucleus.

In order to make the TaTPRPK1 in (a) be convenient to purifying, proteinic N-terminal or C-terminal that can the aminoacid sequence shown in the sequence 1 is formed in by sequence table connect label as shown in table 1.

The sequence of table 1 label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR
Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG
	8	DYKDDDDK
			Strep-tag?II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

Above-mentioned (b) but in the TaTPRPK1 synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.The encoding gene of TaTPRPK1 in above-mentioned (b) can be by the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.

The gene of encoding said proteins also belongs to protection scope of the present invention.

Described gene can be following 1) or 2) or 3) or 4) or 5) dna molecular:

1) sequence 2 is held the dna molecular shown in the 195th to 1679 Nucleotide from 5 ' in the sequence table;

2) sequence 2 is held the dna molecular shown in the 195th to 1690 Nucleotide from 5 ' in the sequence table;

3) dna molecular shown in the sequence 2 in the sequence table;

4) under stringent condition with 1) or 2) or 3) the dna sequence dna hybridization that limits and the dna molecular of coding stress tolerance correlative protein;

5) with 1) or 2) or 3) dna sequence dna that limits has 90% above homology, and the dna molecular of the stress tolerance correlative protein of encoding.

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

CDNA sequence in the sequence 2 is made up of 2112 Nucleotide, and open reading frame is from the 195th to 1679 Nucleotide of 5 ' end.

The recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain described gene all belong to protection scope of the present invention.

Available existing plant expression vector construction contains the recombinant expression vector of described gene.Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, promptly comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor, and the non-translational region of inducing (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as soybean storage protein gene) 3 ' end to transcribe as the Agrobacterium crown-gall nodule all has similar functions.When using described gene constructed recombinant plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter, as the ubiquitin promoter (Ubiquitin) of cauliflower mosaic virus (CAMV) 35S promoter, corn, they can use separately or be used in combination with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce the enzyme of colour-change or the gene of luminophor (gus gene, luciferase genes etc.) as adding the coding that in plant, to express, have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance or anti-chemical reagent marker gene (as anti-weedkiller gene) etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Described recombinant expression vector specifically can be pBT121-TaTPRPK1; Described pBI121-TaTPRPK1 is the recombinant plasmid that the multiple clone site of the described gene of claim 2 being inserted pBI121 obtains, and is preferably the sequence 2 of sequence table is cut the recombinant plasmid that obtains between the recognition site from BamHI and XhoI enzyme that the dna fragmentation shown in the 195th to 1690 Nucleotide of 5 ' end inserts pBI121.

The present invention also protects a kind of method of cultivating transgenic plant, is described gene is imported in the purpose plant, obtains the transgenic plant that resistance of reverse is higher than described purpose plant.Described gene specifically can import in the described purpose plant by described recombinant expression vector.Carry that described expression carrier can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed tissue cultivating is become plant.

Described resistance of reverse specifically can be salt tolerant.

Described purpose plant both can be that monocotyledons also can be a dicotyledons, as Arabidopis thaliana (as the environmental Arabidopis thaliana of Colombia), tobacco (as W38) etc.

Described salt tolerance can be presented as following (I) or at least a (II) or (III):

(I) under salt stress, the chlorophyll content of described transgenic plant is higher than described purpose plant;

(II) under salt stress, the root system of described transgenic plant is longer than described purpose plant;

(III) under salt stress, the root surface area of described transgenic plant is greater than described purpose plant.

Described salt tolerant also can be presented as following (IV) and/or (V):

(IV) under salt stress, the surviving rate of described transgenic plant is higher than described purpose plant;

(V) under salt stress, the root of described transgenic plant grows tall in described purpose plant.

The total length of described gene of increasing or its any segmental primer are to also belonging to protection scope of the present invention.

TaTPRPK1 of the present invention expresses under the inducing of low temperature, high salt, methyl jasmonate, Powdery Mildew, ethene, coerces to express down at arid, high temperature, ABA, Whitfield's ointment and is suppressed.TaTPRPK1 can improve tobacco and the Arabidopis thaliana resistance to NaCl, promotes the growth of root system.TaTPRPK1 albumen can autophosphorylation.Albumen provided by the invention and gene will play an important role in cultivating resistance and resistance of reverse enhanced plant for the degeneration-resistant and anti-retrocorrelation expression of gene of artificial control provides the foundation.

Description of drawings

Fig. 1 is the homology comparison result of TaTPRPK1 and paddy rice, Arabidopis thaliana homologous protein aminoacid sequence.

Fig. 2 is the real-time fluorescence quantitative PCR collection of illustrative plates that TaTPRPK1 gene stress-inducing is expressed; A: methyl jasmonate treatment; B: salt marsh is handled; C: damage to plants caused by sudden drop in temperature processing; D: ethene is handled; E: Whitfield's ointment is handled; F: dormin is handled; G: arid is handled; H: pyroprocessing; I: powdery mildew pathogenic bacteria is handled.

Fig. 3 is that wild-type Arabidopis thaliana and transgenic arabidopsis salt stress are handled the photo after 5 days.

Fig. 4 is that photo and the root length that wild-type Arabidopis thaliana and transgenic arabidopsis salt stress were handled after 2 days compares.

Fig. 5 is that the long comparison of photo, root and the root surface area of wild-type tobacco and transgene tobacco compares.

Fig. 6 is that the blade photo and the chlorophyll content of wild-type tobacco and transgene tobacco compares.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.% among the following embodiment if no special instructions, is the quality percentage composition.

The clone of embodiment 1, TaTPRPK1

One, the separation of mRNA

Xiao Bai wheat (national germplasm resource bank, numbering ZM242) seedling NaCl in tri-leaf period about 10 days handled 2 hours with hydroponics growing, used liquid nitrogen flash freezer, and-80 ℃ of preservations are standby.

Adopt Trizol method (TianGen) to extract the total RNA of wheat leaf blade, the first chain cDNA is synthetic with ThermoScript II XL (AMV).Adopt the synthetic ds cDNA of SMART method, the PCR product carries out 1.0% agarose gel electrophoresis and detects.

Two, the acquisition of TaTPRPK1 full length gene sequence

Method by 5 ' RACE and 3 ' RACE obtains wheat dual specificity protein kinase gene full length sequence.

With the albumen called after TaTPRPK1 albumen shown in the sequence 1 of sequence table, form by 494 amino-acid residues, have conservative dual specificity protein kinase activator district and TPR structural domain.The sequence of TaTPRPK1 is compared on Genabnk, has higher homology (Fig. 1) with dual specificity protein kinases in paddy rice and the Arabidopis thaliana, and do not find the homologous protein gene in wheat, proves that TaTPRPK1 is a new albumen.With the proteic encoding gene called after of TaTPRPK1 TaTPRPK1 gene, its open reading frame is from 5 of the sequence 2 of sequence table ' the 195th-1679 Nucleotide of end.

Embodiment 2, real-time fluorescence quantitative PCR are analyzed the expression characterization of TaTPRPK1

One, coerces processing

Seedling age is 10 days a Xiao Bai wheat seedling, carries out following processing:

(1) arid is handled: the wheat seedling of water planting is taken out the moisture that blots on the root, place on the exsiccant 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, uses liquid nitrogen flash freezer, and-80 ℃ of preservations are standby.

(2) salt marsh is handled: with wheat seedling place 2% by NaCl and Na ₂SO ₄(NaCl and Na in the sodium salt solution of forming ₂SO ₄Mass percent be 3: 2) in, illumination cultivation is taken out material respectively after 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, use liquid nitrogen flash freezer ,-80 ℃ of preservations are standby.

(3) dormin is handled: dormin (ABA) solution that wheat seedling is placed 200 μ M, illumination cultivation takes out and uses liquid nitrogen flash freezer respectively after 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours ,-80 ℃ of preservations are standby.

(4) damage to plants caused by sudden drop in temperature processing: wheat seedling is placed 4 ℃ of incubators, and illumination cultivation takes out and uses liquid nitrogen flash freezer after 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, and-80 ℃ of preservations are standby.

(5) methyl jasmonate treatment: wheat seedling is placed methyl jasmonate (JA) solution of 50 μ M, and illumination cultivation takes out and uses liquid nitrogen flash freezer respectively after 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, and-80 ℃ of preservations are standby.

(6) ethene is handled: wheat seedling places the plastics bag that contains ethene, and illumination cultivation takes out and use liquid nitrogen flash freezer respectively after 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours, 48 hours, and-80 ℃ of preservations are standby.

(7) Whitfield's ointment is handled: wheat seedling is placed Whitfield's ointment (SA) solution of 50 μ M, and illumination cultivation takes out and uses liquid nitrogen flash freezer respectively after 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, and-80 ℃ of preservations are standby.

(8) pyroprocessing: wheat seedling is placed under 42 ℃, and illumination cultivation takes out and uses liquid nitrogen flash freezer respectively after 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, 12 hours, 24 hours, 48 hours, and-80 ℃ of preservations are standby.

(9) powdery mildew pathogenic bacteria is handled: wheat seedling is inoculated the Powdery Mildew bacterial strain, and illumination cultivation is after 3 hours, 6 hours, 12 hours, 2 days, 3 days, 4 days, 5 days, and taking-up is also used liquid nitrogen flash freezer, and-80 ℃ of preservations are standby.

(10) Dui Zhao processing: directly get wheat seedling-80 ℃ frozen (0 hour) in contrast without any processing.

Two, the separation of mRNA

Adopt Quikprep Micro mRNA Purification Kit (Pharmacia) to carry out the separation of mRNA.

Three. reverse transcription is cDNA

Adopting R103-Quant_Reverse_Transcriptase (TIANGEN) is cDNA with the mRNA reverse transcription of purifying.

Four, real-time fluorescence quantitative PCR

According to the TaTPRPK1 sequence, at its variable region design special primer TaTPRPK1RTF and TaTPRPK1RTR (the product size is 135bp).With actin is internal control gene, and primer is actin-2F and actin-2R.

TaTPRPK1RTF：5’-ACATGGCAGAACAGGCTCTC-3’；

TaTPRPK1RTR：5’-CCTTGAGAGCATCTTGAGCTTC-3’。

actin-2F：5’-CTCCCTCACAACAACCGC-3’；

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

Each is coerced TaTPRPK1 and hormone shows response, sees Fig. 2.

Embodiment 3, TaTPRPK1 have improved the salt tolerance of Arabidopis thaliana

One, the structure of recombinant expression vector

1, the clone of TaTPRPK1 gene

To (TaTPRPK1-121F and TaTPRPK1-121R), the primer end introduces BamHI respectively and the XhoI enzyme is cut recognition site according to the sequences Design primer of TaTPRPK1 gene, is template with the cDNA of Xiao Bai wheat, pcr amplification TaTPRPK1.

TaTPRPK1-121F：5’-TCCCCCGGGATGGGCGCCAGGATGTC-3’；

TaTPRPK1-121R：5’-GGACTAGTTCAGTTCATATTCAGCGTCCG-3。

Pcr amplification product carries out 1.2% agarose gel electrophoresis, adopts the band about Agarose Gel DNA Purification KitVer.2.0 (TaKaRa company, Code No.:DV807A) recovery purifying 1.5Kb.

2, the structure of recombinant expression vector

1. cut the PCR product that step 1 reclaims purifying with restriction enzyme BamHI and XhoI enzyme, reclaim enzyme and cut product;

2. cut pBI121 (purchase of Clontech company) with restriction enzyme BamHI and XhoI enzyme, reclaim carrier framework;

3. step enzyme 1. being cut product is connected with step carrier framework 2.;

4. step connection product electric shock is 3. transformed TOP10 bacterial strain (available from tiangen company), 37 ℃ of incubated overnight, the picking positive colony checks order; Sequencing result shows, obtained recombinant plasmid pBI121-TaTPRPK1 (having inserted the sequence 2 of sequence table from the dna fragmentation shown in 5 ' the end 195-1690 position Nucleotide between the BamHI of pBI121 and XhoI restriction enzyme site).

Two, the acquisition of transgenic plant

1, transforms Agrobacterium C58 (purchase of Beijing Baeyer enlightening biotech company) with recombinant plasmid pBI121-TaTPRPK1, obtain the Agrobacterium of recombinating.

2, the Agrobacterium of will recombinating is inoculated in the YEP liquid nutrient medium, and 28 ℃, 3000rpm were cultivated about 30 hours;

3, the bacterium liquid with step 2 goes in the YEP liquid nutrient medium (containing 50 μ g/ml Rifampins), and 28 ℃, 300rpm are cultivated about 14 hours (bacterium liquid OD600 reaches 1.5-3.0);

4, collect thalline, 4 ℃, the centrifugal 10min of 4000g are diluted to OD600 with 10% sucrose (containing 0.02%silwet) and are about 0.8-1.0;

5, with Arabidopis thaliana (the environmental Col-0 of Colombia, the purchase of SALK company) whole strain tips upside down in the container of the bacterium liquid that fills step 4 with flowerpot, flower is soaked about 50s, after immersion finishes, take out flowerpot, be sidelong in pallet, cover black plastic cloth, open plastic cloth behind the 24hr, uprightly place flowerpot, carry out normal illumination cultivation, results T ₁For seed, kantlex screening (concentration is 50 μ g/L kantlex) positive plant.Positive plant is carried out PCR identify, qualification result shows, the transfer-gen plant that obtains (changeing the TaTPRPK1 gene plant).

T ₂T is shown in representative ₁The seed that produces for selfing reaches by the plant that it grew up to T ₃T is shown in representative ₂The seed that produces for selfing and by plant that it grew up to.

Three, change the acquisition of empty carrier control plant

Transform Agrobacterium with plasmid pBI121, obtain the Agrobacterium of recombinating,, obtain changeing the empty carrier adjoining tree, the same step 2 of method with reorganization Agrobacterium-mediated Transformation Arabidopis thaliana.

Four, the salt tolerance of transgenic plant is identified

Respectively with T ₃For transfer-gen plant (WK), T ₃In generation, change the empty carrier adjoining tree and Arabidopis thaliana Col-0 (WT, wild-type) (each 60 strain) carries out the salt tolerance evaluation.Repeated experiments is set three times, results averaged.

The seedling kind that will be in the isometric growth phase is on common MS substratum, and growth is transferred to the MS substratum (NaCl concentration is 200mM) that contains NaCl and gone up continuation cultivation 5 days two days later.Took pictures in the 2nd day and to measure root long, took pictures in the 5th day and add up surviving rate.The 5th day photo is seen Fig. 3, and the surviving rate of Arabidopis thaliana Col-0 is 10%, and the surviving rate of transfer-gen plant is 60%.The 2nd day photo is seen Fig. 4 A, and root length is relatively seen Fig. 4 B, and the root length of transgenic line is about 2 times of Arabidopis thaliana Col-0.The phenotype of changeing the empty carrier adjoining tree is consistent with Arabidopis thaliana Col-0, and surviving rate and Arabidopis thaliana Col-0 do not have significant difference.

Embodiment 4, TaTPRPK1 have improved the salt tolerance of tobacco

One, the structure of recombinant expression vector

Step 1 with embodiment 3.

Two, the acquisition of transgenic plant

2, inoculation reorganization Agrobacterium receives in the LB substratum (card receive the concentration of mycin be 50 μ g/ml) of mycin in containing card, 28 ℃ of cultivations 1-2 days; Be transferred in the 20mL LB substratum, 28 ℃ are continued to be cultured to OD600 about 0.5; Bacterium liquid is mixed with MS substratum equal-volume, obtain soak solution.

3, get aseptic tobacco W38 (available from tobacco institute of the Chinese Academy of Agricultural Sciences) blade and remove vein, be cut into 0.5cm * 0.5cm blade, put into soak solution, soak 10-15min, during constantly jog is several times.

4, take out material, inhale with aseptic paper and remove unnecessary bacterium liquid, 28 ℃ of dark cultivations 2 days in the MS solid medium;

5, use the sterile water wash material, inhale with aseptic paper and remove the thalline of overgrowth, material is transferred in the division culture medium (common MS substratum contains 1mg/L 6-AB and 0.1mg/L IAA), 25 ℃ of light are cultivated down, per two weeks are changed a subculture, to differentiating callus, until sprouting.

6, the bud that will grow to 1cm changes root media (being the 1/2MS substratum) over to and goes up root induction and obtain transfer-gen plant (T ₀Generation)

T ₁T is shown in representative ₀The seed that produces for selfing and by plant that it grew up to.

Three, change the acquisition of empty carrier control plant

Transform Agrobacterium C58 with plasmid pBI121, obtain the Agrobacterium of recombinating,, obtain changeing the empty carrier adjoining tree, the same step 2 of method with the aseptic tobacco W38 of reorganization Agrobacterium-mediated Transformation.

Four, the salt tolerance of transgenic plant is identified

Respectively with T ₁Three strain systems (WK1, WK20, WK68), T for transfer-gen plant ₁In generation, change the empty carrier adjoining tree and wild-type plant (W38) (each 60 strain) carries out the salt tolerance evaluation.Repeated experiments is set three times, results averaged.

The seedling that will be in the isometric growth phase is transferred to the upward growth of MS substratum (NaCl concentration is 200mM) that contains NaCl, as experimental group; The seedling that will be in the isometric growth phase is transferred on the MS substratum and grows, in contrast group.

Take pictures after 5 days and to measure root long, calculate root surface area.Root length, the root surface area mean value of homophyletic system do not see Table 2.Photo is seen Fig. 5 A, and root surface area is relatively seen Fig. 5 B, and root length is relatively seen Fig. 5 C.Genetically modified plant root is obviously huge than wild-type plant.The phenotype of changeing the empty carrier adjoining tree is consistent with the wild-type plant.

Table 2 is the long and root surface area of root of homophyletic system not

After 5 days tobacco is got the 3rd layer of blade, get 5 blades with punch tool, bubble is taken pictures and is measured chlorophyll content in 200mM NaCl salt solution.Chlorophyll content (the chlorophyll gram number that every gram blade contains) sees Table 3, and photo is seen Fig. 6 A, and chlorophyll content is relatively seen Fig. 6 B.

Table 3 is the chlorophyll content of homophyletic system not

	W38	WK1	WK20	WK68
					Mean number	15.29077	64.56527	69.17697	76.7632
Deviation	0.765738	0.331787	1.649356	0.863161

Sequence table

＜110〉Institute of Crop Science, Chinese Academy of Agricultural Science

＜120〉plant stress tolerance correlative protein TaTPRPK1 and encoding gene thereof and application

<130>CGGNARY102247

<160>2

<210>1

<211>494

<212>PRT

＜213〉Triticum wheat (Triticum aestivum L.)

<400>1

Met?Gly?Ala?Arg?Met?Ser?Lys?Ala?Thr?Ser?Cys?Cys?Cys?Leu?Arg?Gly

1 5 10 15

Gln?Leu?His?Gly?Ser?Thr?Arg?Leu?Asp?Asp?Pro?Gly?Ser?Glu?Glu?Asp

20 25 30

Glu?Gln?Gly?Glu?Ala?Tyr?Glu?Leu?Pro?Ala?Phe?Gln?Glu?Tyr?Thr?Phe

35 40 45

Glu?Gln?Leu?Arg?Leu?Ala?Thr?Ala?Gly?Phe?Ala?Val?Glu?Asn?Ile?Val

50 55 60

Ser?Glu?His?Gly?Glu?Lys?Ala?Pro?Asn?Val?Val?Tyr?Lys?Gly?Lys?Leu

65 70 75 80

Asp?Ala?Gln?Arg?Arg?Ile?Ala?Val?Lys?Arg?Phe?Asn?Arg?Ser?Ala?Trp

85 90 95

Pro?Asp?Pro?Arg?Gln?Phe?Leu?Glu?Glu?Ala?Lys?Ser?Val?Gly?Gln?Leu

100 105 110

Arg?Ser?Lys?Arg?Leu?Ala?Asn?Leu?Leu?Gly?Cys?Cys?Cys?Glu?Gly?Asp

115 120 125

Glu?Arg?Leu?Leu?Val?Ala?Glu?Tyr?Met?Pro?Asn?Asp?Thr?Leu?Ala?Lys

130 135 140

His?Leu?Phe?His?Trp?Glu?Thr?Gln?Ala?Met?Lys?Trp?Pro?Met?Arg?Leu

145 150 155 160

Arg?Val?Val?Leu?Tyr?Leu?Ala?Glu?Ala?Leu?G1u?Tyr?Cys?Thr?Ser?Lys

165 170 175

Gly?Arg?Ala?Leu?Tyr?His?Asp?Leu?Asn?Ala?Tyr?Arg?Val?Leu?Phe?Asp

180 185 190

Asp?Asp?Cys?Asn?Pro?Arg?Leu?Ser?Cys?Phe?Gly?Leu?Met?Glu?Asn?Ser

195 200 205

Arg?Asp?Gly?Lys?Ser?Tyr?Ser?Thr?Asn?Leu?Ala?Phe?Thr?Pro?Pro?Glu

210 215 220

Tyr?Met?Arg?Thr?Gly?Arg?Ile?Thr?Pro?Glu?Ser?Val?Ile?Tyr?Ser?Phe

225 230 235 240

Gly?Thr?Leu?Leu?Leu?Asp?Val?Leu?Ser?Gly?Lys?His?Ile?Pro?Pro?Ser

245 250 255

His?Ala?Leu?Asp?Leu?Ile?Arg?Asp?Arg?Asn?Phe?Ser?Met?Leu?Thr?Asp

260 265 270

Ser?Cys?Leu?Glu?Gly?Gln?Phe?Ser?Asn?Glu?Glu?Gly?Thr?Glu?Leu?Val

275 280 285

Arg?Leu?Ala?Ser?Arg?Cys?Leu?His?Tyr?Glu?Pro?Arg?Glu?Arg?Pro?Asn

290 295 300

Val?Arg?Ser?Met?Val?Gln?Ala?Leu?Ala?Pro?Leu?Gln?Lys?Asp?Val?Glu

305 310 315 320

Thr?Pro?Ser?Tyr?Glu?Leu?Met?Asp?Met?Pro?Gln?Ala?Gly?Ala?Ser?Ser

325 330 335

Val?Gln?Ser?Leu?Pro?Leu?Ser?Pro?Leu?Ala?Asp?Ala?Cys?Ser?Arg?Lys

340 345 350

Asp?Leu?Thr?Ala?Ile?His?Glu?Ile?Leu?Glu?Arg?Thr?Gly?Tyr?Lys?Asp

355 360 365

Asp?Glu?Gly?Thr?Ala?Asn?Glu?Leu?Ser?Phe?Gln?Met?Trp?Thr?Asn?Gln

370 375 380

Met?Gln?Asp?Thr?Leu?Thr?Ser?Lys?Lys?Lys?Gly?Asp?Ser?Ala?Phe?Arg

385 390 395 400

Gln?Lys?Asp?Phe?Thr?Thr?Ala?Ile?Asp?Cys?Tyr?Ser?Gln?Phe?Ile?Glu

405 410 415

Val?Gly?Thr?Met?Val?Ser?Pro?Thr?Ile?Tyr?Ala?Arg?Arg?Cys?Leu?Ser

420 425 430

Tyr?Leu?Met?Asn?Asp?Met?Ala?Glu?Gln?Ala?Leu?Ser?Asp?Ala?Met?Gln

435 440 445

Ala?Leu?Val?Ile?Ser?Pro?Thr?Trp?Pro?Thr?Ala?Phe?Tyr?Leu?Gln?Ala

450 455 460

Ala?Ala?Leu?Leu?Ser?Leu?Asp?Met?Glu?Asn?Glu?Ala?Gln?Asp?Ala?Leu

465 470 475 480

Lys?Asp?Gly?Cys?Ala?Gln?Glu?Thr?Ser?Ser?Ser?Ser?Gly?Arg

485 490

<210>2

<211>2112

<212>DNA

＜213〉Triticum wheat (Triticum aestiVum L.)

<400>2

gagaggagag?agccgagaga?gccgaggggc?gagagggagc?gagagagagc?cgagagagat 60

ccaatccaat?ccagcccagt?cccctcccca?gtccccgccc?ccgccgtcgc?cgcgccgctt 120

cgattcgctc?cggttccgcg?cttgctcgct?caggcgaggt?tcggcggcga?gggaggagga 180

ggagggggat?cggaatgggc?gccaggatgt?ccaaggccac?ctcctgctgc?tgcctccgcg 240

ggcagctcca?cggcagcacc?cgcctcgacg?accccggctc?cgaggaggac?gagcaggggg 300

aggcgtacga?gctgccggcg?ttccaggagt?acaccttcga?gcagctgcgc?ctggccacgg 360

ccggcttcgc?cgtggagaac?atcgtgtccg?agcatggcga?gaaggcgccc?aacgtcgtct 420

acaaggggaa?gctcgacgcc?cagcgccgca?tcgccgtcaa?gcgcttcaac?cgctccgcct 480

ggcccgaccc?gcgccagttc?ctggaagaag?ctaaatcagt?tggtcagctc?cggagcaaaa 540

ggttagcaaa?tctgcttggc?tgttgctgcg?aaggtgacga?gagattgctt?gttgcagagt 600

acatgcctaa?cgacacacta?gcaaaacacc?ttttccactg?ggaaacccaa?gcgatgaaat 660

ggcccatgag?attaagggtt?gttctctatc?ttgctgaggc?tttagaatat?tgcaccagta 720

aggggcgtgc?tctctaccat?gatcttaatg?cctacagagt?tctgtttgat?gatgattgta 780

accctagact?ttcatgcttt?ggccttatgg?agaacagccg?ggatggcaaa?agttacagta 840

ccaatctggc?atttactcct?cccgaataca?tgagaactgg?acgtataaca?cctgaaagtg 900

tcatatatag?ctttggcacc?ttgctactgg?acgttcttag?tgggaagcat?attcctccga 960

gccatgccct?tgacctgatt?cgagatcgga?actttagcat?gcttacagac?tcgtgtttag 1020

agggccaatt?ttcaaatgag?gaaggaacag?aactggtacg?attagcttca?agatgcctgc 1080

actatgaacc?ccgagaacgg?ccgaatgtaa?gatctatggt?tcaagcattg?gctcctcttc 1140

agaaagatgt?tgagactcca?tcttacgaac?tgatggatat?gccccaagcc?ggtgcatcat 1200

ctgtccaatc?attgcctctt?tctcctcttg?ctgacgcttg?ttccagaaag?gatctgacag 1260

caatacatga?aattctagaa?aggacagggt?acaaggatga?tgagggaaca?gcaaatgagc 1320

tctcatttca?gatgtggacc?aatcaaatgc?aagatacatt?aacctcaaag?aagaagggtg 1380

acagtgcatt?tcggcaaaag?gattttacta?ctgctattga?ctgttactcc?cagttcattg 1440

aagtcggtac?gatggtgtcc?cccaccattt?atgctcggcg?ttgcctatca?tatctgatga 1500

atgacatggc?agaacaggct?ctcagtgatg?caatgcaagc?gctagtaata?tctccgacat 1560

ggccgactgc?attttacctt?caagctgcag?cgttgctttc?tttagacatg?gagaatgaag 1620

ctcaagatgc?tctcaaggat?ggttgtgccc?aagagacaag?tagcagcagc?ggacgctgaa 1680

tatgaactga?tttcttgtct?aggaaatgca?cataattcca?ggtgctcgct?tgaacaaaat 1740

catttggaat?ggacgaagca?gctgtttttc?atctgtcaac?tttggccaag?tgttctttga 1800

gcttcttatg?atttgcgtgc?atgagacaac?gtcgtccctc?ctccgtatgt?aaatatggaa 1860

caaagcgaca?acagagacca?ggaaagagat?gccatataca?ttgaagggtg?aataaccccc 1920

gtagacagga?aattttttgt?ggaagcgaaa?aatccgtctc?ggttcctccc?accgaagaaa 1980

gggcgaaaag?aagagagatt?gttgatgtac?attatttgta?aacctgtaca?gtttttcctt 2040

ttcggagcag?gatcgtcttt?ggctgaatgg?ttgccaaaaa?ttctcagttt?ctattaaaaa 2100

aaaaaaaaaa?aa 2112

Claims

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

2. coding claim 1 described proteic gene.

3. gene as claimed in claim 2 is characterized in that: it is for following 1) or 2) or 3) or 4) or 5) dna molecular:

3) dna molecular shown in the sequence 2 in the sequence table;

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

5. recombinant expression vector as claimed in claim 4 is characterized in that: described recombinant expression vector is pBI121-TaTPRPK1; Described pBI121-TaTPRPK1 is the recombinant plasmid that the multiple clone site of the described gene of claim 2 being inserted pBI121 obtains, and is preferably the sequence 2 of sequence table is cut the recombinant plasmid that obtains between the recognition site from BamHI and XhoI enzyme that the dna fragmentation shown in the 195th to 1690 Nucleotide of 5 ' end inserts pBI121.

6. a method of cultivating transgenic plant is that claim 2 or 3 described genes are imported in the purpose plant, obtains the transgenic plant that resistance of reverse is higher than described purpose plant.

7. method as claimed in claim 6 is characterized in that: claim 2 or 3 described genes import in the described purpose plant by claim 4 or 5 described recombinant expression vectors; Described resistance of reverse is a salt tolerant.

8. method as claimed in claim 7 is characterized in that: described purpose plant is a tobacco, is preferably tobacco bred W38; Described salt tolerance is presented as following (I) or at least a (II) or (III):

(II) under salt stress, the root length of described transgenic plant is greater than described purpose plant;

9. method as claimed in claim 7 is characterized in that: described purpose plant is an Arabidopis thaliana, is preferably the environmental Arabidopis thaliana of Colombia; Described salt tolerant is presented as following (IV) and/or (V):

(V) under salt stress, the root length of described transgenic plant is greater than described purpose plant.

10. total length or its any segmental primer of amplification claim 2 or 3 described genes are right.