CN102924582B - Plant-adversity-resistance related protein TaNAC67 as well as coding gene and application thereof - Google Patents

Abstract

The invention discloses a plant-adversity-resistance related protein as well as a coding gene and an application of the plant adversity-resistance related protein. The protein can be selected from protein (a) which is composed of amino acid sequences shown by sequence 2 in a sequence table, or protein (b) which is derived from the protein (a) and is formed by substituting and/or deleting and/or adding one or more amino acid residues for the amino acid sequences of the sequence 2 in the sequence table, and is related to plant-adversity-resistance. Experiments prove that the gene can be guided into the plant cells to improve the plant adversity resistance such as drought resistance water saving, salt resistance or cold resistance. The gene and the application of the gene provided by the invention have important practical significance to the culturing of the new drought-resistant water-saving, salt-resistant or cold-resistant varieties.

Description

Plant adversity resistance related protein TaNAC67 and encoding gene thereof and application

Technical field

The present invention relates to a kind of plant anti-adversity associated protein TaNAC67 and encoding gene and application.

Background technology

Drought and water shortage is the serious problems that Global Agriculture production faces, and is also the important factor of restriction China Grain Production Development.The cultivation of staple food crop wheat needs a large amount of water, 1 ton of about 500-700m of wheat water requirement of the average every production of China ³.Whole world developing country has 6,000 ten thousand hectares of wheat cultivations at least at dry farmland, but its yield level only has the 10%-50% under irrigation conditions.So development resisting drought saving water wheat breed, to improve the water use efficiency of crop, both can increase output, can alleviate again the contradiction of shortage of water resources.

The Crop Improvement drought resistance is significant for improving agricultural productive force, is subject to the great attention of countries in the world, and the great special project of genetically modified crops rearing new variety of China's startup in the recent period is exactly best real example.The drought resistance mechanism of research plant, clone's gene related to drought tolerance, by genetically engineered Crop Improvement drought resistance, be an effective way of cultivating the drought-resistant crops new variety.although the heredity of drought resistance of crop basis is complicated, the clone, transform the obvious new variety difficulty that improves of anti-drought gene acquisition drought resistance larger, but the joint efforts through numerous scientists, obtained certain progress, many successful examples have been emerged in large numbers, as (Wheat LEA genes such as Cheng, PMA80 and PMA1959, enhancedehydration tolerance of transgenic rice (Oryza sativaL.), Molecular Breeding, 2002, 10:71-82) the lea protein gene Introduced into Rice of wheat, the drought resisting of transfer-gen plant, salt tolerance is significantly improved, (the Over-expression of an Arabidopsis δ-OAT gene enhances saltand drought tolerance intransgenic rice such as WU, Chinese Science Bulletin:2003,48:2594-2600) change the δ of Arabidopis thaliana-OAT gene in paddy rice overexpression, in transfer-gen plant, proline content obviously increases, and drought resisting and salt tolerance obviously strengthen, (the OsDREB genes in rice such as Dubouzet, Oryza sativaL., encode transcription activators that function in drought-, high-salt-andcold-responsive gene expression, Plant J, 2003,33:751-763) after paddy rice transcription activating protein gene OsDREB was imported to Arabidopis thaliana, transfer-gen plant had showed strong drought resistant, anti-salt and cold resistant property.Hu etc. import to the OsNAC1 gene in paddy rice, the drought resistance of transgenic progeny is obviously strengthened, and do not affect offspring's output (Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhancesdrought resistance and salt tolerance in rice, Hu etc. 2006, Proc Natl Acad SciUSA, 10:71-82).

At present, the gene related to drought tolerance of having cloned mainly wraps two large classes, the first kind is functional gene, this genoid comprises the synthesis related genes such as low molecular weight soluble sugar, amino acid and small protein, and Cell protection is exempted from injured enzyme, as the proline synthesis relative enzyme gene, comprise Δ' pyrroline 5 carboxlate synthetase gene P5CS and PVAB2, pyrroles's beautiful jade-5-carboxylate reductase gene P5CR etc.; Late embryo generation Abundant protein (LEA), wheat lea protein gene (The molecular basis of dehydration tolerance in plants, Ingram etc. 1996, AnnuRev Plant Physiol Plant Mol Biol, 47:377-403).

Equations of The Second Kind is regulatory gene, comprise the gene that various participation water stress signals transmit, comprise that mainly (1) participates in synthetic crucial enzyme (the Plant responses to water deficit of the signaling molecules such as ABA, ethene, Bray1997, Trends in Plant Science, 2:48-54); (2) phosphoprotein phosphatase, as protein phosphatase 2A and 2C etc., they participate in transmission (the Disruption of a guard cell-expressed proteinphosphatase 2A regulatory subunit of ABA signal, RCN1, confers absci sic acidinsensitivityin Arabidopsis, Kwak etc. 2002, Plant Cell, 14:2849-2861; The Arabidopsis ABSCISICACID-INSENSITIVE2 (ABI2) and ABI 1genes encode homologous protein phosphatases2C involved in abscisic acid aignal transduction, Leung etc. 1997, Plant Cell, 9:759-771; The ABI1 and ABI2 protein phosphatase 2C act in a negative feedbackregulatory loop of the abscisic acid signalling pathway, Merlot etc. 2001, PlantJ, 25:295-303; Mutational analysis of protein phosphatase 2C involved inabscisic acid signal transduction in higher plants, Sheen 1998, Proc Natl AcadSci USA, 95:975-980).(4) protein phosphatase kinases, plant protein kinase family very, mainly comprise mitogen activated protein kinase (MAPK) (Abscisic a ci d induces mitogen-activated protein kinaseactivation in barley aleurone protoplasts, Knetsch etc. 1996, Plant Cell, 8:1061-1067), calcium dependent protein kinase (CDPK) (An abscisic acid-activated andcalciumindependent protein kinasa from guard cells of fava bean, Li etc. 1996, Plant Cell, 8:2359-2368, CDPK-mediated signaling pathways:specificity andcross-talk, Sauer etc. 2004, J Exp Bot, 55:181-188, Ca ²⁺dependent protein kinaseand stress signal transduction in plant, Sheen 1996, Science, 274:1900-1902) and the non-fermentation related protein kinase of sucrose (SnRK).(3) transcription factor (The Arabidopsis homeobox geneAtHB-7 is induced by water deficit and by abscisic acid, Soderman etc. 1996, Plant J, 10:375-381), as DREB(Wang etc. 2008, Plant Mol Bio, 67:589-602, Stress-inducible DREB2A transcription factor from Pennisetum glaucum is aphosphoprotein and its phosphorylation negatively regulates its DNA-bindingactivity, Agarwal etc. 2007, Mol Genet Genomics, 277:189-198, GmDREB2, a soybeanDRE-binding transcription factor, conferred drought and high-salt tolerance intransgenic plants, Chen etc. 2007, Biochem Biophys Res Commun, 353:299-305, Identification of cold-inducible downstream genes of the ArabidopsisDREB1A/CBF3transcript ional factor us ing two micro-array systems, Maruyama etc. 2004, Plant J, 38:982-993, OsDREB genes in rice, Oryza sativa L., encodetranscription activators that function in drought-, high-salt-andcold-responsive gene expression, Dubouzet etc. 2003, Plant J, 33:751-763), NF-YB1(Plant nuclear factor Y (NF-Y) B subunits confer drought tolerance and lead toimproved corn yields on water limited acres, Nelson etc. 2007, Proc Natl Acad SciUSA, 104:16450-16455), HD-ATART(Activated expression of an ArabidopsisHD-START protein confers drought tolerance with improved root system andreduced stomatal density, Yu etc. 2008, Plant Cell, 20:1134-1151), OsNAC1(Overexpressing a NAM, ATAF, and CUC (NAC) transcription factor enhancesdrought resistance and salt tolerance in rice, Hu etc. 2006, Proc Natl Acad SciUSA, 10:71-82) etc.

NAC (NAM, ATAF and CUC) transcription factor is one of distinctive transcription factor superfamily of plant.Such transcription factor N end is the DNA binding domains of high conservative, and its C end is the very large transcriptional activation domain of variation (Comprehensive analysis of NAC family genes in Oryza sativa and Arabidopsisthaliana, Ooka etc. 2003, DNA Res, 10:239-247).At present, in model plant Arabidopis thaliana and paddy rice, found more than 100 family member (Systematic sequence analysis and identification oftissue-specific or stress-responsive genes of NAC transcription factor familyin rice, Fang etc. 2008, Mol Genet Genomics, 280:547-563; Comprehensive analysisof NAC fami ly genes in Oryza sativaand Arabidopsis thaliana, Ooka etc. 2003, DNA Res, 10:239-247; Transcription factor families in Arabidopsis:majorprogress and outstanding issues for future research, Qu etc. 2006, Curr Opin PlantBiol, 9:544-549).Many evidences show, the NAC transcription factor plays an important role in the growth and development process of plant, as embryo, flower, secondary wall formation, leaf senile, (the The no apical meristem geneof Petunia is required for pattern format ion in embryos and flowers and isexpressed at meristem and primordia boundaries such as root growth, Souer etc. 1996, Cell, 85:159-170; NAC transcription factors NST1and NST3 regulate pod shattering in a partiallyredundant manner by promoting secondary wall formation after the establishmentof tissue identity, Mitsuda etc. 2008, Plant J, 56:768-778; SND1, a NAC domaintranscription factor, is a key regulator of secondary wall synthesis in fibersof Arabidopsis, Zhong etc. 2006, Plant Cell, 18:3158-3170; Two NAC domaintranscription factors, SND1and NST1, function redundantly in regulation ofsecondary wall synthesis in fibers of Arabidopsis, Zhong etc. 2007, Planta, 225,1603-1611; AtNAP, a NAC family transcription factor, has an important role inleaf senescence, Guo etc. 2006, Plant J, 46:601-612; The Arabidopsis NACtranscription factor VNI2 integrates abscisic acid signals into leafsenescence via the COR/RD genes, Yang etc. 2011, Plant Cell, 23:2155-2168; SoybeanNAC transcription factors promote abiotic stress tolerance and lateral rootformation in transgenic plants, Hao etc. 2011, Plant J, 68:302-313; AtNAC2, atranscription factor downstream of ethylene and auxin signaling pathways, isinvolved in salt stress response and lateral root development, He etc. 2005, Plant J, 44:903-916; TaNAC2, a NAC-type wheat transcription factor conferringenhanced multiple abiotic stress tolerances in Arabidopsis, Mao etc. 2012, J ExpBot, 63:2933-2946; Arabidopsis NAC1 transduces auxin signal downstream of TIR1to promote lateral root development, Xie etc. 2000, Genes Dev, 14:3024-3036).

In the recent period, increasing evidence shows, the NAC transcription factor participates in the replying of various adverse circumstances and abiotic stress, and comprises disease, arid, high salt, damages to plants caused by sudden drop in temperature, anoxic etc.In Arabidopis thaliana, it is found that ANAC019, ANAC055 and ANAC072 can be combined with the upstream promoter of ERD1 gene, thereby strengthen drought resistance (the Isolationand functional analysis of Arabidopsis stress-inducible NAC transcriptionfactors that bind to a drought-responsive cis-element in the early responsiveto dehydration stress 1promoter of Arabidopis thaliana, Tran etc. 2004, Plant Cell, 16:2481-2498).AtAF1, homologous gene HvNAC6 in AtAF2 and barley has negative effect (The transcription factor ATAF2 represses the expression ofpathogenesis-related genes in Arabidopsis in drought resisting and disease resistance response process, Delessert etc. 2005, Plant J, 43:745-757; The HvNAC6transcription factor:a positive regulator of penetrationresistance in barley and Arabidopsis, Jensen etc. 2007, Plan tMolBiol, 65:137-150; A novel drought-inducible gene, ATAF1, encodes a NAC family protein thatnegatively regulates the expression of stress-responsive genes in Arabidopsis, Lu etc. 2007, Plant Mol Biol, 63:289-305).in paddy rice, overexpression SNAC1/OsNAC1 can strengthen drought resistance (the Overexpressing a NAM of paddy rice, ATAF, and CUC (NAC) transcriptionfactor enhances drought resistance and salt tolerance in rice, Hu etc. 2006, Proc Natl Acad SciU USA, 103:12987-12992), and overexpression SNAC2/OsNAC6, OsNAC5, OsNAC045, resistance (the Characterization oftranscription factor gene SNAC2 conferring cold and salt tolerance in rice of OsNAC063 transgenic plant to multiple abiotic stress, Hu etc. 2008, Plant Mol Biol, 67:169-181, Functional analysis of a NAC-typetranscription factor OsNAC6 involved in abiotic and biotic stress-responsivegene expression in rice, Nakashima etc. 2007, Plant J, 51:617-630, The abioticstress-responsive NAC-type transcription factor OsNAC5 regulatesstress-inducible genes and stress tolerance in rice, Takasaki etc. 2010, Mol GenetGenomics, 284:173,183, Tolerance to various environmental stresses conferredby the salt-responsive rice gene ONAC063 in transgenic Arabidopsis, Yokotani etc. 2009, Planta, 229:1065-1075, Overexpression of a NAC transcription factorenhances rice drought and salt tolerance, Zheng etc. 2009, Biochem Biophys ResCommun, 379:985-989).The NAC gene GmNAC20 of overexpression soybean can strengthen salt tolerance and resistance to cold (the Soybean NAC transcription factors promote abiotic stress toleranceand lateral root formation in transgenic plants of Arabidopis thaliana, Hao etc., 2011, Plant J, 68:302-313).The researchist in wheat, find TaGRAB1 and TaGRAB2 can with particle virus RepA protein binding, thereby (the GRAB proteins that copies that suppresses virus, novel members of the NAC domainfamily, isolated by their interaction with a geminivirus protein, Xie etc. 1999, PlantMol Biol, 39:647-656).Xia etc. (2010) find, TaNAC4 and TaNAC8 participate in multiple biology and abiotic stress coerced to reaction (Characterization of a novel wheat NAC transcriptionfactor gene involved in defense response against stripe rust pathogen infectionand abiotic stresses, Xia etc. 2010, Mol Biol Rep, 37,3703-3712; TaNAC8, a novelNAC transcription factor gene in wheat, responds to stripe rust pathogeninfection and abiotic stresses, Xia etc. 2010, Physiol Mol Plant Pathol, 74,394-402).Overexpression TaNAC2 and TaNAC69 can strengthen the resistance (TaNAC2 of plant to multiple adverse circumstance, aNAC-type wheat transcription factor conferring enhanced multiple abioticstress tolerances in Arabidopsis, Mao etc. 2012, J Exp Bot, 63:2933-2946; Overexpression of TaNAC69 leads to enhanced transcript levels of stressup-regulated genes and dehydration tolerance in bread wheat, Xue etc. 2011, MolPlant, 4:697-712).

Summary of the invention

An object of the present invention is to provide a kind of plant anti-adversity associated protein and encoding gene thereof.

Plant anti-adversity associated protein provided by the present invention, derive from common wheat (Triticum aestivum L.), for following (a) or the albumen (b):

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

(b) by the aminoacid sequence of sequence in sequence table 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant to plant stress-resistance by (a) derivative protein.

In order to make the albumen in (a) be convenient to purifying, N end or C end that can the protein that the aminoacid sequence shown in sequence 2 forms in by sequence table connect label as shown in table 1.

The sequence of table 1. label

Label	Residue	Sequence
			Poly-Arg	5-6(is 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 (a) but or the albumen synthetic (b), also can first synthesize its encoding gene, then carry out biological expression and obtain.The encoding gene of the albumen in above-mentioned (b) can by by sequence in sequence table 1 from 5 ' DNA sequence dna shown in end the 93rd to 986 bit bases in the codon of one or several amino-acid residue of disappearance, 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 table 1.

The replacement of one or several amino-acid residue, replacement and/or interpolation in the aminoacid sequence of above-mentioned albumen, have plenty of because abiogenous polymorphism variation causes, such as the difference of the species of the biology by obtaining protein, individuality etc., causes; Have plenty of by induced mutationss such as site-directed mutagenesis, random mutagenesises and process and cause.

Encoding gene provided by the present invention is following 1), 2), 3) or 4) gene:

1) its nucleotide sequence be in sequence table sequence 1 from the DNA molecular shown in 5 ＇ end 93-986 position deoxyribonucleotides;

2) its nucleotide sequence is the DNA molecular shown in sequence 1 in sequence table;

3) under stringent condition with 1) or 2) the DNA sequence dna hybridization that limits and the DNA molecular of the described resistance relevant protein of encoding;

4) with 1) or 2) DNA sequence dna that limits has the DNA molecular of 90% above homology and the described resistance relevant protein of encoding.

Above-mentioned stringent condition is, at 6 * SSC, in the solution of 0.5%SDS, under 65 ° of C, hybridizes, and then uses 2 * SSC, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.

In sequence table, sequence 1 is comprised of 1098 deoxyribonucleotides, comprises the ORF district of 5 ' UTR, the 894bp of 92bp, 3 ' UTR of 112bp; The open reading frame of this gene be in the 894bp(sequence table sequence 1 from 5 ' end 93-986 position Nucleotide), 297 amino acid of encoding (in sequence table shown in sequence 2).

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

Available existing plant expression vector construction contains the recombinant expression vector of gene of the present invention.

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

The method of cultivation adversity resistant plant provided by the present invention, be that above-mentioned arbitrary described encoding gene is imported in the purpose plant, cultivates and obtain adversity resistant plant.

Can adopt ordinary method that described encoding gene is imported in the purpose plant, particle bombardment for example, the high voltage electric perforation method, liposome method, bacterium transforms or transfection etc.Concrete operations in the present invention are, first, by in gene delivery system, obtain recombinant expression vector, then recombinant expression vector is imported in Agrobacterium, obtain containing the restructuring Agrobacterium of gene of the present invention, then by Agrobacterium, gene are imported in the purpose plant.

Above-mentioned adversity resistant plant is drought-resistant and/or high salt tolerance/low temperature resistant plant.

The inventive method to monocotyledons or dicotyledons all can, monocotyledons specifically can be wheat, dicotyledons specifically can be Arabidopis thaliana.

Experiment showed, in gene transfered plant of the present invention, can promote the growth of transgenic plant root system.Improved the resistance of plant, as drought resistance and/or salt resistance and/or resistance to cold.Under drought condition, the survival rate that changes the plant of gene of the present invention over to is 30-60%, and the survival rate that does not change the wild-type of gene of the present invention over to and change the control plant of empty carrier over to only has 5-8%; Under the high-salt stress condition, the survival rate of plant can reach 35-65%, and the survival rate that does not change the wild-type plant of gene of the present invention over to only has 25%; Under the frozen stress condition, the survival rate of plant can reach 18-38%, and the survival rate that does not change the wild-type plant of gene of the present invention and empty carrier contrast over to only has 8-13%.Gene pairs unifacial leaf of the present invention, dicotyledons are all applicable simultaneously.Therefore, gene of the present invention and application thereof have great importance to cultivation resisting drought saving water, anti-salt, cold-resistant new crop varieties, are suitable for applying.

The accompanying drawing explanation

Fig. 1 is TaNAC67 and difference between different plant NAC member sequences.

Fig. 2 is that TaNAC67 is subjected to the expression after water stress, high salt, low temperature and ABA process.

Fig. 3 is the relative expression quantity of TaNAC67 in different Arabidopis thaliana strains.

Fig. 4 is drought resisting, salt tolerant and the frost resistance qualification result of transgenic arabidopsis.

Embodiment

Experimental technique in following embodiment, if no special instructions, be ordinary method.

Percentage composition in following embodiment, if no special instructions, be the quality percentage composition

The invention will be further described below in conjunction with specific embodiment, but the present invention is not limited to following examples.

In following embodiment, if no special instructions, be ordinary method.

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

The separation of the encoding gene TaNAC67 of resistance relevant protein in embodiment 1, wheat

One, the separation of the encoding gene TaNAC67 of resistance relevant protein

1, build the cDNA library of wheat, according to document (with improved Cap-trapper method structure Ae.speltoides cDNA library, Mao Xinguo etc. 2005, Acta Genetica Sinica, 32(8): method 811-817) is carried out, and is specific as follows described:

(1) total RNA extracts and the mRNA purifying, with TRIZOL, extracts wheat (No. 1, the exhibition of laying down, National crop genebank of China) the total RNA of different tissues organ, uses oligo(dT) Mierocrystalline cellulose separation and purification mRNA.

(2) first chain cDNA's is synthetic: get 10ug mRNA and mix with primer I (table 1), after sex change, add the synthetic reagent of the first chain cDNA, when temperature is raised to 40 ℃, add ThermoScript II, when proceeding to 40 minutes, reaction adds primer II (the first chain synthetic primer is as shown in table 2).For obtaining more full-length cDNAs, when the first chain is synthetic, in reaction system, add trehalose and mountain plough sugar alcohol; For the length of restriction poly (A) tail, so that large scale sequencing substitutes traditional single primer oligo (dT) 18 with mix primer.Reaction is removed carbohydrate with the CTAB-UREA method after finishing, precipitation cDNA/RNA.

Table 2. the first chain cDNA synthetic primer

(3) sodium periodate oxidation upwards walks in reaction tubes and adds sodium periodate solution, and oxidation RNA uses the glycerine termination reaction.

(4) cDNA/RNA of the centrifugal collection sodium periodate oxidation of biotin labeling, after cleaning, dry dissolving again, add vitamin H (Biotin) marking fluid (purchased from Sigma) of fresh configuration, and 23 ℃ of incubation 14～16h, use the sodium citrate solution termination reaction.

(5) RNase I digestion is after sodium periodate oxidation, and mRNA 5 ' and 3 ' holds adjacent glycol group on last bit base ribose to be oxidized into the dialdehyde group, they can with the vitamin H combination.When the coated magnetic bead of later-stage utilization Streptavidin separated full-length cDNA, the vitamin H of mRNA 3 ' end also can be combined with magnetic bead.For obtaining the cDNA that 5 ' end is complete, must specifically the vitamin H of 3 ' end mark be removed.Eukaryote mRNA 3 ' end poly(A) length is generally at 100～250bp, when synthetic the first chain cDNA, the length of poly (A) is limited in to 16 bases, therefore in the cDNA/RNA complex body, mRNA 3 ' holds poly(A) will exist with the form of strand, therefore can be with the RNase I by its special removing.

(6) full-length cDNA catch and strand cDNA discharges and first to use the tRNA that pollutes without the DNA magnetic bead (Dynalbead M-280) of blockading, under room temperature, allow cDNA/RNA and magnetic bead in conjunction with 20mi n, with NaOH-EDTA wash-out cDNA/RNA.

(7) the terminal enzyme (DNA) tailing is collected strand cDNA, after sex change, adds terminal transferase reaction reagent, and 37 ℃ were reacted 9 minutes, termination reaction.

The synthetic collection strand cDNA of (8) second chain cDNA, with synthetic the second chain cDNA of LA-Taq.After question response finished, electrophoresis, reclaimed the cDNA greater than 1kb.

(9) restriction enzyme Bsa I belongs to two class restriction enzymes, and its restriction enzyme site just in time is in first base place in recognition site downstream, and enzyme cuts and there is no base specific, but to the cytosine methylation sensitivity of recognition site.The DNA that cuts through Bsa I enzyme will produce the cohesive terminus that 4 bases are outstanding.According to these characteristics, when design of primers, introduced Bsa I, EcoR I and Xho I site, in the first strand primer, be wherein Bsa I and Xho I site, in the second strand primer, be Bsa I and EcoR I site.By taking these measures, only with the Bsa I, cDNA is carried out to single enzyme and cut, just can realize the directed cloning of cDNA.

(10) connection, packing and Insert Fragment detect: the purpose cDNA fragment after the collection classification is dissolved in ddH again ₂In O, detect cDNA concentration, after determining the concentration of cDNA, get appropriate cDNA and be connected and spend the night with carrier UniZAP II (Stratagene).After packing, infect Host Strains XL1-Blue, detect titre.

(11) plasmid extraction and sequencing output, then repetitive proteins enzyme K digestion, after the step such as phenol/chloroform extraction amplified library, get a certain amount of amplification library rapid for the phasmid ring, finally cDNA is placed in to the ethanol precipitation and spends the night.Cyclisation, detect the phasmid titre, and the phasmid of finally getting after appropriate cyclisation infects the SOLR host cell.

(12) the host cell spread plate that phasmid was infected, 37 ℃ of overnight incubation.The picking positive colony, in 96 well culture plates, extracts plasmid, order-checking immediately, builds the Wheat Full-length cDNA database.

From wheat, to suppress poor cDNA library (Analysis of gene expression profile responedto water stress in wheat (the Triticum aestivum L) seedling that subtracts, Pang etc. 2007, Acta Agronomica Sinica 32, the sequence that obtains 333-336) is source sequence, search Wheat Full-length cDNA database, obtain candidate clone, and order-checking obtains target clone's full length sequence, its sequence is in sequence table shown in sequence 1, and the sequence total length is 1098bp.

Shown in analytical sequence 1, the structure of gene shows, it is that the 5 ' UTR(92bp), the 93-986 position Nucleotide is open reading frame (894bp) from 5 ' end 1-92 position Nucleotide, and 987-1098 position Nucleotide is 3 ' UTR(112bp).

The aminoacid sequence of this genes encoding, as shown in sequence in sequence table 2, is comprised of 297 amino-acid residues.

The open reading frame table of sequence and the sequence of public database are compared, find that this gene encoding production has NAC transcription factor family member's characteristics, simultaneously with the BdNAC67 of false bromegrass and the ZmNAC67 of corn, has very high similarity, therefore by the unnamed gene shown in sequence 1 in sequence table of the present invention, be TaNAC67, by the albumen of its coding (in sequence table shown in sequence 2) called after TaNAC67.

Take protein TaNAC 67 as source sequence, the protein library in search NCBI public database, download the higher sequence of property similarly.Carry out sequence alignment, comparison result shows the TaGRAB1 of protein TaNAC 67 of the present invention and wheat, BdNAC67 and ZmNAC67 Relationship Comparison nearly (Fig. 1, Bd, Brachypodium distachyon(false bromegrass); Zm, Zea mays(corn)).

Two, the expression characteristic of plant stress-resistance genes involved TaNAC67

(1) plant stress-resistance genes involved TaNAC67 is experiment material to the situation of replying of different environment stresses with Drought resistant Wheat (drought is selected No. 10, National crop genebank of China).

Select full seed, Drought resistant Wheat seed of the same size (drought is selected No. 10), be placed in illumination box, 20 ℃, 12h/d cultivation, water planting to a leaf wholeheartedly, then carries out the environment stress processing.The concrete grammar that environment stress is processed is as described below:

1) water stress (drought stress; With the PEG6000 simulating drought, coerce environment): remove the moisture in culture dish, (osmotic potential is the-0.5MPa) aqueous solution to add PEG-6000;

2) high-salt stress (NaCl): remove the moisture in culture dish, add the 250mM NaCl aqueous solution;

3) low temperature stress (LT): directly culture dish is moved to 4 ℃ of illumination boxs and cultivate;

4) exogenous aba treatment (ABA): adopt 50 μ M ABA solution to spray until blade is all moistening.

Respectively 1)-4) Different stress 0,1,2,3,6,12,24,48 and the 72h that process gather blade, liquid nitrogen flash freezer ,-70 ℃ save backup.Contrast adopts deionized water to cultivate always.

With TRIZOL, extract the total RNA of wheat, with synthetic the first chain cDNA(Invitrogen of MMLV reverse transcription test kit), adopt the method for real-time quantitative PCR (Real-time Quantitative PCR, qRT-PCR) to detect the response condition of gene TaNAC67 to various environment stresses.With the Tubulin gene of constitutive expression, as internal reference, designed the primer (table 3) of qRT-PCR.

The primer of table 3.qRT-PCR

The formula that proposes according to Livak and Schmittgen calculates (Analysis of relative geneexpression data using real-time quantitative PCR and the 2 (Delta Delta C (T)) Method.Methods, 2001,25,402-408): the expression amount of TaNAC67 gene under 4 kinds of processing is N times of contrast, N=2 ^{-△ △ TT}, △ △ TT=(CT _{(Target, Timex)}-CT _{(Tubulin, Timex)})-(TC _{(Target, Time0)}-CT _{(Tubulin, Time0)}).

Wherein, the implication of CT value is: the cycle number that the fluorescent signal in each reaction tubes experiences while arriving the thresholding of setting.When PCR circulates in the cycle number that arrives CT value place, just entered the real index amplification phase (logarithmic phase), this moment, slight error was not yet amplified, so the circulation ratio of CT value is fabulous, be amplification in same template different time amplification or same asynchronism(-nization) pipe, the CT value that obtains is constant.

Time x represents different treatment time points; The zero point that time 0 representative is processed; CT _{(Target, Timex)}For passing through Stress treatment x during the time, the expression amount of TaNAC67 gene in wheat; CT _{(Tubulin, Timex)}For passing through Stress treatment x during the time, the expression amount of Tubulin gene in wheat; C _{T (Target, Time0)}When not starting Stress treatment, the expression amount of TaNAC67 gene in wheat; C _{T (Tubulin, Time0)}When not starting Stress treatment, the expression amount of Tubulin gene in wheat.

3 repetitions are established in experiment, and result is taken the mean, and result as shown in Figure 2.Relative expression quantity is the N value.Result shows, TaNAC67 participates in the replying of PEG, NaCl, low temperature and ABA, but to the pattern difference of different Stress responses.In Fig. 2, PEG represents drought stress, and NaCl represents high-salt stress, and LT represents low temperature stress, and ABA represents exogenous aba treatment.

Embodiment 2, the application of gene TaNAC67 in Arabidopis thaliana

One, build transgenosis TaNAC67 Arabidopis thaliana

(GI:506685) (The small take pPZP211 as initial carrier, versatile pPZP family ofAgrobacterium binary vectors for plant transformation, Hajdukiewicz etc. (1994), Plant Mol Biol, 25:989-994) (Institute of Crop Science, Chinese Academy of Agricultural Science), between the polyclone position of pPZP211 Sal I and PSt I site, insert GFP(BAG13014) open reading frame, finally obtain binary vector pPZP211-GFP(TaSnRK2.4, an SNF1-type serine-threonine protein kinase of wheat (Triticum aestivumL.) confers enhanced multi-stress tolerance in Arabidopsis, Mao etc. 2010, Journal of experimental botany, 61:683-696).

According to gene TaNAC67 full length sequence, design primer: upstream primer F1:

5 '-CTCTAAGCTTGATGGCGGCGGCGGAGCG-3'(Hind III site), downstream primer R1:

5 '-CTCTGGATCCGTAACGGCCGCCAGTGTGCTG-3'(BamH I site), wherein downstream primer 3 ' end is positioned at the upstream of gene terminator codon.

Extract common wheat (Triticum aestivum L.) drought and select the mRNA of No. 10, take 5'-CGGAGAAGCAGGAAGCGGCAAT-3' and 5'-GTACCGGCTGGCAATGTATCATT-3' as primer, with the method for RT-PCR, obtain the full-length cDNA of gene TaNAC67 in wheat; Take the full length cDNA sequence of gene TaNAC67 as template, with above-mentioned primer, adopt high-fidelity enzyme Pfu amplification target gene, amplified production is carried out to enzyme with restriction enzyme Hind III and BamH I and cut, recovery target gene fragment; With restriction enzyme Hind III and BamH I enzyme, cut binary vector pPZP211-GFP, reclaim the purpose carrier segments; The target gene fragment of recovery is connected with the purpose carrier segments, screens, obtain containing the positive recombinant vectors pPZP211-TaNAC67/GFP of sequence 1 83-986 position Nucleotide (being the open reading frame of gene TaNAC67) in ordered list.In pPZP211-TaNAC67/GFP, take CaMV 35S as promotor, TaNAC67 and GFP gene are positioned at its downstream, and this recombinant vectors imports in plant, can express the TaNAC67-GFP fusion rotein.

Utilize agriculture bacillus mediated method pPZP211-TaNAC67/GFP to be forwarded in Arabidopis thaliana to the Arabidopis thaliana that obtains transgenosis TaNAC67.With the MS Screening of Media transfer-gen plant that is added with kantlex, the expression of TaNAC67-GFP fusion rotein in observation Arabidopis thaliana young root under fluorescent microscope, screening obtains the positive and turns the pPZP211-TaNAC67/GFP plant.

According to the power of fluorescence, select transgenic line breeding that fusion protein expression is higher, add generation, obtain the transgenosis pure lines, then according to the function of the method validation TaNAC67 in following two.Take the wild-type Arabidopis thaliana that do not change any carrier over to, change empty carrier pPZP211-GFP over to Arabidopis thaliana as contrast.

Two, the transgenic arabidopsis expression amount detects

The sowing of transgenic arabidopsis kind (is contained to 0.8% agar) on the MS substratum, then be placed under 22 ℃, the condition of 12h illumination/d and cultivate, each strain is collected 20 Arabidopsis thaliana Seedlings, with Trizol, extract the total RNA of Arabidopis thaliana, with synthetic the first chain cDNA of MMLV reverse transcription test kit (purchased from Invitrogen), adopt the method for qRT-PCR to detect the expression amount (the detection primer of gene TaNAC67 in Table 3) of target gene TaNAC67 in different transgenic lines.(primer is as F:5 '-GAGGCCTCGTGTGGTCGCTTTGT-3 ' take Arabidopis thaliana Tubulin gene; R:5 '-GCCCAGTTGTTACCCGCACCAGA-3 ') compare, take the expression level of target gene in the minimum transgenic line of expression amount (L1) as benchmark (expression amount is decided to be 1), calculate TaNAC67 relative expression quantity in each transgenic line.

Turn TaNAC67 expression amount detected result in the pPZP211-TaNAC67/GFP Arabidopis thaliana: in 6 transgenic lines of random choose, expression amount is followed successively by from high to low: L4 > L2 > L6 > L5 > L3 > L1.(Fig. 3, L1～L6 are 6 and turn TaNAC67 gene strain).

Three, the resistance of transgenic arabidopsis detects

By cultivating soil (vermiculite and ratio humous are 1:1) in the rectangular plastic charging tray, water to after saturated, the Arabidopsis thaliana Seedlings of 7 ages in days (comprise the contrast of transgenic line, wild-type, turn the empty carrier contrast) is transplanted in polypots, and the control water planting is supported under 22 ℃, 12h illumination/d, relative humidity 70% condition.

(1) Identification of Drought

Control water to wild-type Arabidopis thaliana and the Arabidopis thaliana that turns empty carrier seriously wilted, and rehydration was then taken a picture respectively on the 2nd day after first 3 days of rehydration and rehydration.

(2) salt resistance is identified

With the NaCl solution pouring of 250mM, observe the degeneration-resistant situation of transfer-gen plant and also take a picture in good time.

3 repetitions are established in test, and result is taken the mean.

(3) Identification of Cold Tolerance

Claim equivalent by cultivating soil in the square plastic magazine, water to after saturated, the Arabidopsis thaliana Seedlings of 7 ages in days (comprise transgenic line, wild-type contrast, turn the empty carrier contrast) is transplanted to (every box 4 young plants) in polypots, the control water planting is supported under 22 ℃, 12h illumination/d, relative humidity 70% condition, after 3 weeks, seedling is placed in the refrigerated tank of-10 ℃ and processes 1.0h, then at 15 ℃ of recovery 24h, then cultivate under normal operation, after 3 days, observe statistical results.

3 repetitions are established in test, and result is taken the mean.

Identification of Drought survival rate: the survival rate average out to 30-60% of transfer-gen plant, and wild-type and empty carrier contrast Arabidopis thaliana is respectively 8% and 5%(Fig. 4 A, 4D, WT represents wild-type, and L1～6 are 6 and turn TaNAC67 gene strain, and GFP is the contrast that turns the pZP211/GFP carrier).

Salt resistance is identified survival rate: the survival rate average out to 35-65% of transfer-gen plant, and wild-type Arabidopis thaliana and the contrast survival rate that changes empty carrier over to be respectively 25% and 20%(Fig. 4 B, 4D).

The Identification of Cold Tolerance survival rate: the survival rate average out to 18-38% of transfer-gen plant, and the average survival rate of the contrast of wild-type Arabidopis thaliana and carrier be respectively 8% and 13%(Fig. 4 C, 4D).

Fig. 4 A is Identification of Drought survival rate photo result; Fig. 4 B is that salt resistance is identified survival rate photo result; Fig. 4 C is Identification of Cold Tolerance survival rate photo result; Fig. 4 D is three kinds and coerces appraising datum bar chart result (wherein, in three cylindricality results of each strain, the cylindricality order represents the result that Identification of Cold Tolerance, Identification of Drought and salt resistance are identified from left to right).

Claims (7)

1. albumen, the protein that is formed by the aminoacid sequence shown in sequence in sequence table 2.

2. the encoding gene of the described albumen of claim 1.

3. encoding gene according to claim 2, it is characterized in that: described encoding gene is following 1) or 2) gene:

1) its nucleotide sequence be in sequence table sequence 1 from the DNA molecular shown in 5 ＇ end 93-986 position deoxyribonucleotides;

2) its nucleotide sequence is the DNA molecular shown in sequence 1 in sequence table.

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

5. the application of albumen claimed in claim 1 in the plant of cultivating the resistance raising;

Described adversity resistant plant is drought-resistant and/or salt tolerant and/or cold-resistant plant;

Described plant is wheat or Arabidopis thaliana.

6. a method of cultivating adversity resistant plant, be that the described encoding gene of claim 2 or 3 is imported in the purpose plant, cultivates and obtain adversity resistant plant;

Described adversity resistant plant is drought-resistant and/or salt tolerant and/or cold-resistant plant;

Described purpose plant is wheat or Arabidopis thaliana.

7. method according to claim 6 is characterized in that: described encoding gene imports in described purpose plant by recombinant expression vector claimed in claim 4.

Images