CN106337041A - Plant stress-tolerance associated protein and coding gene and application thereof - Google Patents
Plant stress-tolerance associated protein and coding gene and application thereof Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
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- C12Y306/01003—Adenosine triphosphatase (3.6.1.3)
Abstract
The invention discloses a plant stress-tolerance associated protein and a coding gene and an application thereof. The protein is shown as the following (a) or (b): (a) a protein consisting of amino acid residue sequences shown in SEQ ID No:1 in a sequence table; and (b) a protein formed by that amino acid residue sequences shown in SEQ ID No:1 in a sequence table are replaced and/or deleted and/or added by one or more amino acid residues, and is related to plant stress tolerance. The protein provides an economic, rapid and effective approach for increasing the plant stress tolerance, the protein has wide application and market prospect in field of agriculture, and provides an important candidate gene for improving crop stress tolerance for gene engineering.
Description
Technical field
The invention belongs to genetic engineering field, it is related to a kind of plant stress tolerance correlative protein and its encoding gene and application.
Background technology
The abiotic stress such as arid, saline and alkaline, extreme temperature cause to the growth of crop and seriously threaten, and plant is produced
Raw a series of morphology, the impact of Physiology and biochemistry state and molecular level, cause vine growth and development slow, seriously
When even cause irreversible injury, even result in whole plant and shift to an earlier date death, greatly affect the yield of crop.Alive
In the range of boundary, abiotic stress is the main cause causing crop failure, occupies the 50% of crop failure every year on average.
Under the conditions of environment stress, plant as a complicated biosystem, often produce a series of forms, Physiology and biochemistry,
The change of the levels such as molecule, gene and metabolite, thus produce certain resistance or patience to adverse circumstance.
Because plant is perphyton, in the face of poor environment when adverse circumstance can not be hidden by shift position.In order to survive,
Plant evolution goes out a series of raising environment stress resistances or patience strategy.About plant stress-resistance, resistance of reverse research always
It is the study hotspot in botany field.By the resistance of traditional breeding technique improvement plant, resistance of reverse difficulty relatively
Larger it is impossible to obtain more excellent degeneration-resistant, resistance to inverse kinds well.And the development with Protocols in Molecular Biology,
And the further investigation to plant stress-resistance, resistance to inverse molecular mechanism, molecular level degeneration-resistant (resistance to inverse) genetic engineering obtains heavy
Big progress.At present, importing external source resistance of reverse gene using genetic engineering means such as transgenic to plant has become improvement plant
One of new way of thing resistance of reverse.Study plant stress-resistance, resistance to inverse molecular regulation network and turn out more excellent drought-enduring
Kind has boundless prospect and highly important meaning.
Under the conditions of environment stress, plant can be by a series of physiology, Biochemical changes and metamorphosis, to adverse circumstance
Stress produces certain adaptation.Vacuole is the coated balloon-shaped structure of the distinctive film of plant cell, accounts in ripe cell
More than the 90% of cell volume.Under the conditions of environment stress, plant adjusts Premeabilisation of cells pressure by vacuole, remains thin
Intracellular water balance, accumulation and storage nutrient, multiple metabolite and noxious substance, are issued in environment stress condition
Material energy metabolism, the balance of g and D.V-atpase (vacuole v- type atp synthase) is important on vacuole
Enzyme, has substantial connection with the substance transportation across tonoplast, and v-atpase relies on the energy transfer matter that hydrolysis atp produces
Son, thus producing the electrochemical gradient of cross-film and adjusting the ph value of intraor extracellular, promotes the na in Cytoplasm+Etc. transferring to
In vacuole and accumulate, eliminate na+Murder by poisoning to cell.Therefore, v-atpase is during the degeneration-resistant Stress responses of plant
There is extremely important effect.V-atpase is made up of multiple subunits, and space structure assumes " bulb " structure.Prominent
Part outside film is referred to as v1, and film interior part is referred to as v0.About 8 kinds subunit compositions of v1 part, according to molecular size range
It is respectively designated as a～h, v0 part is about made up of three kinds of subunits.The e subunit of wherein v1 part be one have abundant
The hydrophilic protein of charged residues, has pivotal role in the assembling process of v-atpase albumen.
Content of the invention
It is an object of the invention to provide a kind of plant stress tolerance correlative protein and its encoding gene and application.
Protein provided by the present invention, entitled Sorghum vulgare Pers. vacuole atp synthase e protein subunit (sbatpase-e),
From Sorghum vulgare Pers. (sorghum bicolor l.), it is following (a) or (b):
A () aminoacid sequence is the protein of sequence 1 in sequence table；
The aminoacid sequence of b protein that (a) is limited by () through one or several amino acid residues replacement and/
Or disappearance and/or interpolation, and the protein related to plant stress tolerance.
For the ease of described protein purification, the amino acid residue sequence of sequence 1 can form in by sequence table
The amino terminal of protein or carboxyl terminal connect upper label as shown in the table.
Table: the sequence of label
| Label | Residue | Sequence |
| poly-arg | 5-6 (usually 5) | rrrrr |
| poly-his | 2-10 (usually 6) | hhhhhh |
| flag | 8 | dykddddk |
| strep-tag ii | 8 | wshpqfek |
| c-myc | 10 | eqkliseedl |
Protein in above-mentioned (b) can synthetic, also can first synthesize its encoding gene, then carry out biological expression and obtain
Arrive.The encoding gene of the protein in above-mentioned (b) can be by by the dna sequence shown in sequence in sequence table 2
Lack the codon of one or several amino acid residues, and/or the missense mutation carrying out one or several base pairs.
The nucleic acid molecules of code for said proteins fall within protection scope of the present invention.
Described nucleic acid molecules can be dna, such as cdna, genome dna or restructuring dna；Described nucleic acid molecules
Can also be rna, such as mrna, hnrna or trna etc..
In one embodiment of the invention, described nucleic acid molecules are specially the gene of code for said proteins；Described base
Because can be for following 1) to 4) in arbitrary described dna molecule:
1) nucleotides sequence is classified as the dna molecule of the 152-757 position of sequence 2 in sequence table；
2) nucleotides sequence is classified as the dna molecule of sequence 2 in sequence table；
3) under strict conditions with 1) or 2) dna of the dna molecule hybridization that limits and code for said proteins divides
Son；
4) with 1) or 2) or 3) the dna molecule that limits has more than 90% homology and code for said proteins
Dna molecule.
Above-mentioned stringent condition can be with 6 × ssc, the solution of 0.5%sds, hybridizes at 65 DEG C, then with 2 × ssc,
0.1%sds and 1 × ssc, 0.1%sds respectively wash film once.
Wherein, sequence 2 is made up of 1016 nucleotide, and 152-757 position is orf, sequence in polynucleotide
Protein shown in 1.
Recombinant vector containing above-mentioned nucleic acid molecules, expression cassette, transgenic cell line or recombinant bacterium fall within the present invention's
Protection domain.
Described recombinant vector can be recombinant expression carrier, alternatively recombinant cloning vector.
Described recombinant expression carrier can use existing plant expression vector construction.Described plant expression vector includes double base agriculture
Bacillus carrier and can be used for carrier of plant micropellet bombardment etc., such as pgreen0029, pcambia3301,
Pcambia1300, pbi121, pbin19, pcambia2301, pcambia1301-ubin or other are spread out
Plant expression vector.Described plant expression vector also can comprise 3 ' end untranslated regions of exogenous gene, that is, comprise to gather
Adenosine acid signal and the dna fragment of any other participation mrna processing or gene expression.Described polyadenylation signals
Bootable polyadenylic acid is added to 3 ' ends of mrna precursor.During using described gene constructed recombinant expression carrier,
Any enhancement mode, composing type, organizing specific type or inducible promoter can be added before its transcription initiation nucleotide,
Such as cauliflower mosaic viruses (camv) 35s promoter, ubiquitin gene ubiquitin promoter (pubi), the side of body
Urgent inducible promoter rd29a etc., they be can be used alone or are used in combination with other plant promoters；Additionally,
Using the present invention gene constructed recombinant expression carrier when, it is also possible to use enhancer, including translational enhancer or transcription increase
Hadron, these enhancer regions can be atg start codon or neighboring region start codon etc., but must be with
The reading frame of coded sequence is identical, to ensure the correct translation of whole sequence.Described translation control signal and initiation codon
The source of son is extensive, can be natural or synthesis.Translation initiation region can come from transcribing
Beginning region or structural gene.For the ease of being identified to transgenic plant cells or plant and being screened, can be to weight used
Group expression vector is processed, and such as adds the coding that can express in plant can produce enzyme or the light emitting compound of color change
The gene of thing, the antibiotic marker thing with resistance or anti-chemical reagent marker gene etc..Also any selection can be not added with
Property marker gene, directly with adverse circumstance screen transformed plant.
In an embodiment of the present invention, the promoter starting described genetic transcription in described recombinant expression carrier is specially
35s promoter.
More specifically, described recombinant expression carrier be pcambia3301 carrier restriction enzyme site bglii and
The recombiant plasmid obtaining after dna fragment shown in sequence 3 in insertion sequence table between bste.
Described expression cassette is by the promoter that can start described gene expression, described gene, and transcription terminator group
Become.
Described transgenic cell line is non-propagating materialss.
Described protein or described nucleic acid molecules or described recombinant vector, expression cassette, transgenic cell line or recombinant bacterium exist
Application in (a1) or (a2) as follows falls within protection scope of the present invention:
(a1) regulate and control plant stress tolerance；
(a2) plant variety that selection-breeding resistance of reverse improves.
In the present invention, described regulation and control plant stress tolerance is embodied in: in described plant body, if the table of described protein
Within the specific limits, expression is higher for the amount of reaching, then the resistance of reverse of described plant is stronger；If described protein expression amount
Lower, then the resistance of reverse of described plant is weaker.
In the present invention, the method for the plant variety that described selection-breeding resistance of reverse improves, specifically may include described protein
The step that the higher plant of expression is hybridized as parent.
It is a further object to provide a kind of method cultivating the transgenic plant that resistance of reverse improves.
The method cultivating the transgenic plant that resistance of reverse improves provided by the present invention, comprising:
A) import the encoding gene of described protein in recipient plant, the transgenic obtaining expressing described encoding gene is planted
Thing；
B) obtain compared with described recipient plant from step a) gained transgenic plant, the transgenic that resistance of reverse improves
Plant.
Described gene can be for following 1) to 4) in arbitrary described dna molecule:
1) nucleotides sequence is classified as the dna molecule of the 152-757 position of sequence 2 in sequence table；
2) nucleotides sequence is classified as the dna molecule of sequence 2 in sequence table；
3) under strict conditions with 1) or 2) dna of the dna molecule hybridization that limits and code for said proteins divides
Son；
4) with 1) or 2) or 3) the dna molecule that limits has more than 90% homology and code for said proteins
Dna molecule.
Above-mentioned stringent condition can be with 6 × ssc, the solution of 0.5%sds, hybridizes at 65 DEG C, then with 2 × ssc,
0.1%sds and 1 × ssc, 0.1%sds respectively wash film once.
Described encoding gene specifically can be imported in described purpose plant by any of the above-described described recombinant expression carrier, obtains
Described transgenic plant.Specifically can turn by using ti plasmid, ri plasmid, plant viral vector, direct dna
Described recombinant expression carrier is turned by the conventional biology methods such as change, microinjection, conductance, agriculture bacillus mediated, particle gun
Change plant cell or tissue, and the plant tissue of conversion is cultivated into plant.
In above-mentioned application or method, at least one during described resistance of reverse is all concretely following: drought resistance, salt resistance
Property and aba toleration.Described resistance of reverse is presented as the resistance of reverse in Their Seed Germinating Period and/or seedling stage.
Described plant is dicotyledon or monocotyledon.Described dicotyledon is crucifer, such as intends south
Mustard.Described monocotyledon is grass, such as Sorghum vulgare Pers..
The primer pair of the total length or its arbitrary fragment that expand the encoding gene of described protein falls within the protection model of the present invention
Enclose.
The present invention is that the resistance of reverse improving plant provides an economy, fast and effectively approach；The present invention is in agricultural
Field will have wide application and market prospect, provides important time for genetic engineering Crop Improvement resistance of reverse in future
Select gene.
Brief description
Fig. 1 is the structure flow chart of recombinant expression carrier pcambia3301-35s::sbatpase-e.
Fig. 2 is that bacterium solution pcr of recombinational agrobacterium agl0/pcambia3301-35s::sbatpase-e identifies electrophoresis
Figure.Wherein, swimming lane m is dna molecular weight standard；Remaining swimming lane is positive restructuring Agrobacterium
agl0/pcambia3301-35s::sbatpase-e.
Fig. 3 is that t3 identifies for the pcr of transgenic arabidopsis homozygous lines, and pcr the primer is vector primer, mesh
Fragment total length 1500bp.Wherein, swimming lane m is dna molecular weight standard；Swimming lane 1 and 2 is respectively the positive
T3 is for transgenic arabidopsis homozygous lines fox30.3 and fox30.4.
Fig. 4 be sprouting stage wildtype Arabidopsis thaliana and t3 for transgenic line fox30.3 and fox30.4 150mm,
Phenotype under the nacl process of 175mm.
Fig. 5 be sprouting stage wildtype Arabidopsis thaliana and t3 for transgenic line fox30.3 and fox30.4 at 0.2 μm
The phenotype coming off under acid treatment.
Fig. 6 be wildtype Arabidopsis thaliana and t3 for transgenic line fox30.3 150mm nacl and 250mm
Phenotype after 1 week for the treatment with mannitol.
Specific embodiment
Experimental technique used in following embodiments if no special instructions, is conventional method.
Material used, reagent etc. in following embodiments, if no special instructions, all commercially obtain.
Xinjiang drought-enduring sorghum variety xgl-1: Grain Crops Inst., Xinjiang Agricultural Academy.Be recorded in " Lu Min. beautiful
The clone of rice zmsnac1 and Sorghum vulgare Pers. sbsnac1 gene and functional analyses. the Chinese Academy of Agricultural Sciences, crop germplasm provides
Source is learned, and 2013, thesis for the doctorate " literary composition, the public can obtain at applicant, can only be used to repeat present invention experiment.
Pdonr222 carrier: invitrogen Products, it is also recorded in " copt6is a plasma simultaneously
membrane transporter that functions in copper homeostasis in arabidopsis and is a novel
Target of squamosa promoter binding protein-like 7 " one literary composition, the public can obtain at applicant,
Can only be used to repeat present invention experiment.
Pmd18-t carrier: takara, catalog no.d101a.
Pcambia3301 plasmid: be recorded in " a maize stress-responsive nac transcription factor,
Zmsnac1, confers enhanced tolerance to dehydration in transgenic arabidopsis, plant cell
Rep, 2012 " literary composition, the public can obtain at applicant, can only be used to repeat present invention experiment.
Agrobacterium agl0: be recorded in " agriculture bacillus mediated gna gene transformation Elite Maize Inbred Lines and transgenic corns
Pest-resistant Disease-resistance Analysis, Shandong University Ph.D. Dissertation in 2005 " one literary composition, the public can obtain, only at applicant
Can be used for repeating present invention experiment.
Arabidopsiss (columbia-0): be recorded in " a maize stress-responsive nac transcription factor,
Zmsnac1, confers enhanced tolerance to dehydration in transgenic arabidopsis, plant cell
Rep, 2012 " literary composition, the public can obtain at applicant, can only be used to repeat present invention experiment.
The discovery of embodiment 1, sbatpase-e protein subunit and its encoding gene
By Sorghum vulgare Pers. total length cdna library construction on expression vector, extensive arabidopsis thaliana transformation, through a large amount of drought resistings
Character identification and Molecular Detection find a new albumen from the drought-enduring sorghum variety xgl-1 of Xinjiang, are named as
Sbatpase-e protein subunit.
The aminoacid sequence of sbatpase-e protein subunit is sequence 1 in sequence table.By sbatpase-e protein subunit
Encoding gene be named as sbatpase-e gene, its cdna is as shown in the sequence 2 of sequence table.
Extract the total rna of blade of the Xinjiang drought-enduring sorghum variety xgl-1 of Osmotic treatment, and reverse transcription is cdna.
Using cdna as template, pcr amplification is carried out using primer f and r, sequencing is carried out to gained pcr product.
Result shows the sequence of pcr product just shown in sequence 2 in sequence table.
Primer f:5 '-acaagtttgtacaaaaaagtt-3 '；
Primer r:5 '-gattacaccaagtgcgggtgt-3 '.
Embodiment 2, the acquisition of transfer-gen plant and resistance of reverse identification
First, the acquisition of transfer-gen plant
1st, the structure of recombinant expression carrier
The structure flow chart of recombinant expression carrier is shown in Fig. 1.
(1) extract total rna of the Xinjiang drought-enduring sorghum variety xgl-1 of Osmotic treatment, and reverse transcription is cdna.
Using cdna as template, pcr amplification is carried out using primer f and r (sequence is ibid) and obtains sequence 2 in sequence table
Shown sbatpase-e gene (can certainly artificial synthesized sequence 2), being reacted by gateway (bp) will
Sbatpase-e gene (sequence 2) restructuring imports pdonr222 carrier, and gained recombiant plasmid is named as
pdonr222-sbatpase-e.
(2) add the recognition sequence of restriction enzyme site bgl and bste at gateway sequence two ends respectively, obtain
(hold from 5 ' and be followed successively by restriction enzyme site bgl, attr1, ccdb, attr2 to 3 ' ends to dna fragment shown in sequence 4
With restriction enzyme site bste), with pmd18-t carrier after obtain carrying out bgl and bste enzyme action after recombiant plasmid,
Reclaim and purification digestion products (size is about the purpose band of 2430bp).
(3) use restricted enzyme bglii and bste double digestion pcambia3301 plasmid, reclaim carrier framework
(about 9250bp), the gus fragment between bglii and bste is cut off.
(4) the carrier endonuclease bamhi of the enzyme action recovery product of step (3) and step (2) is connected, obtain matter of recombinating
Grain pcambia3301-attr1-ccdb-attr2.Described recombiant plasmid pcambia3301-attr1-ccdb-attr2
Structure be described as: will in pcambia3301 plasmid be located at restriction enzyme site bglii and bste between small fragment
The recombiant plasmid that (gus gene) obtains after replacing with dna fragment shown in sequence 4 in sequence table.
(5) sequence in the recombiant plasmid pdonr222-sbatpase-e that will be obtained in step (1) by lr reaction
Recombiant plasmid pcambia3301-attr1-ccdb-attr2 is obtained in sbatpase-e gene shown in 2 and step (4)
In ccdb gene occur restructuring exchange, obtain recombiant plasmid pcambia3301-35s::sbatpase-e.Described
Recombiant plasmid pcambia3301-35s::sbatpase-e structure is described as follows: by pcambia3301 plasmid
Small fragment (gus gene) between restriction enzyme site bglii and bste replaces with sequence table shown in sequence 3
The recombiant plasmid obtaining after dna fragment.
2nd, the acquisition of transgenic plant and identification
1) step one is built the recombinant expression carrier pcambia3301-35s::sbatpase-e conversion agriculture of gained
Bacillus agl0, obtains recombinational agrobacterium.To recombinational agrobacterium, the identification of bacterium solution pcr is carried out using primer f1 and r1.
The pcr identification of positive Agrobacterium is as shown in Fig. 2 obtain the purpose band that size is about 695bp.
Primer f1:5 '-atgaatgacaccgatgtagccaa-3 ' (the 152-173 position of sequence 2)；
Primer r1:5 '-the ttaggctgcagtctggcca-3 ' (reverse mutual of the 828-846 position of sequence 2
Complementary series).
The plasmid extracting the positive recombinational agrobacterium of pcr identification sends to sequencing, and result shows that extracted plasmid is just
(sequencing shows to be located at restriction enzyme site in pcambia3301 plasmid pcambia3301-35s::sbatpase-e
Small fragment (gus gene) between bglii and bste replaces with dna fragment (sequence shown in sequence 3 in sequence table
Row 3 are held from 5 ' and are followed successively by attb1, sbatpase-e, attb2 to 3 ' ends) after the recombiant plasmid that obtains), illustrate be
Positive restructuring Agrobacterium, is named as agl0/pcambia3301-35s::sbatpase-e.
2) recombinational agrobacterium agl0/pcambia3301-35s::sbatpase-e is cultivated, 28 DEG C (200rmp)
Shaking to od600 value is 1.2-1.4.Collects thalline, with appropriate permeabilization buffer (formula: 1/2ms culture medium
Middle addition 5% (5g/100ml) sucrose) fully suspension thalline to bacterium solution od600 value is 0.8 about, to infiltration buffering
The adsorbent silwet l-77 of final concentration of 0.02% (volume fraction) of addition in bacterium solution after liquid suspension, obtains agriculture
Bacillus bacterium solution.
3) ready arabidopsiss (columbia-0) petal is completely soaked in step 2) the Agrobacterium bacterium solution of gained
In infect about 1min, be shaken gently for inflorescence so that inflorescence is fully immersed in bacterium solution, will plant after 16 DEG C of dark processing 24h
Strain is transferred to growth under normal growing conditions and is repeated conversion 1-2 time, mixes and receives t0 for arabidopsiss seed.
4) t0 is laid in nutritive cube for arabidopsiss seed, each nutritive cube about spreads 1000 seeds, emerges
After proceed by Osmotic treatment, when Arabidopsis plant most in nutritive cube are dead, the plant of survival in each nutritive cube
Strain carries out rehydration process when only having 1-5 strain, and then individual plant harvests seed, obtains t1 for transgenic line.
To t1 for drought-enduring positive transgenic strain individual plant sowing, in the ms containing cremart (ppt) (7mg/l)
On flat board identify (due to cremart resistant gene bar being carried on pcambia3301 carrier), and screen t2,
For the transgenic line (segregation ratio identification) of homozygosis, parent's strain that all offsprings are respectively provided with cremart resistance is pure to t3
Close strain.Adopt with primer f2 and r2 of pcambia3301 vector backbone sequence design to by cremart resistance
The homozygous lines that identification obtains carry out Molecular Identification.Simultaneously using the arabidopsiss (columbia-0) of non-transgenic as open country
Raw type comparison (wt).
Primer f2:5 '-tctccactgacgtaagggat-3 ' (the 2579-2598 position of sequence 3)；
Primer r2:5 '-the ttaggtttacccgccaatat-3 ' (reverse mutual of the 4019-4038 position of sequence 3
Complementary series).
Wherein, the pcr qualification result of t3 generation positive arabidopsiss homozygous lines is as shown in figure 3, obtain expected size about
Purpose band for 1460bp.And wild type control does not obtain purpose band through amplification.Turn from identification positive t3 generation
Randomly select two strains in gene arabidopsiss homozygous lines, be designated as fox30.3 and fox30.4 respectively, for follow-up
Resistance of reverse is identified.
Experiment arranges unloaded comparison it may be assumed that proceeding to in Agrobacterium agl0 with reference to as above method simultaneously
(pcambia3301-35s:: δ gus carrier is to use restriction enzyme to pcambia3301-35s:: δ gus carrier
The skeleton after enzyme bglii and bste double digestion pcambia3301 plasmid, the size of gained being about 9250bp carries
Gained is connected after body end-filling), gained recombinational agrobacterium is named as agl0/pcambia3301-35s:: δ
gus.With further reference to as above method, recombinational agrobacterium agl0/pcambia3301-35s:: δ gus is imported and intend south
Mustard (columbia-0), obtains t0 for arabidopsiss seed.Obtain t3 through selfing and as above cremart resistance screening
For homozygous lines.Also pass through the pcr amplification of as above primer f2 and r2, the size of gained pcr product is about
444bp, consistent with expected resultss.
2nd, the resistance of reverse identification of transfer-gen plant
1st, germination period stress experiment
The kind for homozygous transgenic arabidopsiss strain fox30.3 and fox30.4 for the t3 obtaining will be screened through step one
Son, and proceed to the t3 of empty carrier for the seed of homozygosis arabidopsiss strain and wildtype Arabidopsis thaliana (columbia-0)
(wt) seed of strain carries out disinfection, respectively by its program request containing variable concentrations nacl (150mm, 175mm)
In ms culture medium, it is processed with compareing with the ms culture medium of aba (0.2 μm), each strain point 35
Grain seed, setting 5 repetition, the seed avoid light place on flat board in 4 DEG C of vernalization 2-4 days, through the seed of vernalization
It is put into sprouting (22 DEG C of 16h illumination/18 DEG C 8h dark, relative air humidity under normal growing conditions between culture
40%-50%), count the seed germination rate of each process after 7 days.
Result shows:
(1) toleration of nacl is analyzed
In comparison ms culture medium, two separate transgenic strains of fox30.3 and fox30.4 and wild type control
Germination rate no significant difference between strain (wt).And on the ms flat board containing 150mm, 175mm nacl,
Fox30.3 and fox30.4 transgenic line shows obvious salt tolerance compared with WT strain (wt).In 150mm
Under nacl existence condition, about 33% wildtype Arabidopsis thaliana (wt) seed is sprouted, and fox30.3 and fox30.4
The germination rate of this 2 transgenic lines respectively reaches 72% and 56%, is significantly higher than wild type (wt) arabidopsiss
Germination rate (p < 0.05).Under 175mm nacl existence condition, about 2.7% wildtype Arabidopsis thaliana (wt) is planted
Son is sprouted, and the germination rate of this 2 transgenic lines of fox30.3 and fox30.4 respectively reaches 25.7% and 20.0%,
It is significantly higher than the germination rate (p < 0.05) of wildtype Arabidopsis thaliana (wt) seed.It can be seen that, seed sprouts the stage,
The overexpression of sbatpase-e gene significantly improves the resistance (as Fig. 4) to salt stress for the transgenic line.
Either in comparison ms culture medium, or on the ms flat board containing 150mm or 175mm nacl,
The germination rate proceeding to the t3 of the empty carrier seed for homozygosis arabidopsiss strain is all basic with wildtype Arabidopsis thaliana (wt)
Unanimously, no difference of science of statistics.
(2) toleration of aba is analyzed
Two separate transgenic strains of fox30.3 and fox30.4 and wildtype Arabidopsis thaliana (wt) are coerced in aba
Under germination rate be all inhibited to a certain extent.Under conditions of 0.2 μm of aba exists, fox30.3 and
The seed that this 2 transgenic lines of fox30.4 have 70%-76% is sprouted, and the germination rate of wild type (wt) seed
Only 28%, both significant differences (p < 0.05) (Fig. 5).Result of study display aba sprouts to transgenic line
Inhibitory action be substantially less than WT lines (wt).
On the ms flat board containing 0.2 μm of aba, the t3 proceeding to empty carrier is for the seed of homozygosis arabidopsiss strain
Germination rate is basically identical with wildtype Arabidopsis thaliana (wt), no difference of science of statistics.
2nd, seedling stage stress experiment
By 4 -day-old through step one t3 that obtains of screening for homozygous transgenic arabidopsiss strain fox30.3 and
Fox30.4, and proceed to the t3 of empty carrier for homozygosis arabidopsiss strain and wildtype Arabidopsis thaliana (columbia-0) strain
The seedling of system (wt) is transferred to normal ms culture medium respectively, contains 150mmol l-1Nacl or 350mmol l-1
On the ms plating medium of Mannitol, flat board is disposed vertically, and growth of seedling counts each group seedling blade after one week is big
Little, root length, lateral root number isophenous character.Each 12 seedling of repetition of each strain, 3 repetitions of setting.
Result shows:
(1) (salt-resistance) is analyzed to the toleration of nacl
Wild type (wt) and fox30.3 the and fox30.4 transgenic line of one week are grown on normal ms culture medium
Tie up to leaf blade size, root length, there is no notable difference in lateral root number isophenous character.Containing 150mmol l-1
Fox30.3 the and fox30.4 transgenic line growing in the ms culture medium of nacl and the growth of WT strain are all
Significantly suppressed, but the impact to WT lines for the nacl is larger, its blade is compared with fox30.3 and fox30.4
Transgenic line substantially diminishes (p < 0.05), and root system substantially shortens compared with fox30.3 and fox30.4 transgenic line,
Wherein transgenic line is 4.2cm, the only 3.4cm of WT strain, significant difference (p < 0.05) (Fig. 6).
Either in normal ms culture medium, or containing 150mmol l-1On the ms flat board of nacl, turn
The t3 entering empty carrier is all basically identical with wildtype Arabidopsis thaliana for the phenotype of homozygosis arabidopsiss strain plant, no statistics
Difference.
(2) (drought resistance) is analyzed to the toleration of Mannitol
Containing 350mmol l-1In the ms culture medium of Mannitol, fox30.3 and fox30.4 transgenic line and
The growth of WT strain (wt) is also all significantly suppressed, but fox30.3 and fox30.4 transgenic is planted
Strain is more healthy and stronger compared with WT lines (wt), and transfer-gen plant root system is more flourishing, transgenic line tip of a root number
Dramatically increase compared with wild type, transgenic line average tip of a root number is 4.8, and wild type average tip of a root number is 3.5
(p<0.05).In addition, transfer-gen plant aerial partss still behave as green, and WT lines (wt) are on the ground
Part has occurred and that browning (Fig. 6).
Containing 350mmol l-1On the ms flat board of Mannitol, the t3 proceeding to empty carrier is for homozygosis arabidopsiss strain
The phenotype of plant is basically identical with wildtype Arabidopsis thaliana (wt), no difference of science of statistics.
In sum it is seen that sbatpase-e gene provided by the present invention is favorably improved the salt tolerance of plant, resistance to
Drought and the toleration to aba.
Claims (10)
1. protein, is following (a) or (b):
A () aminoacid sequence is the protein of sequence 1 in sequence table；
The aminoacid sequence of b protein that (a) is limited by () through one or several amino acid residues replacement and/
Or disappearance and/or interpolation, and the protein related to plant stress tolerance.
2. the nucleic acid molecules of protein described in coding claim 1.
3. nucleic acid molecules according to claim 2 it is characterised in that: described nucleic acid molecules be coding right will
Seek the gene of protein described in 1；Described gene is following 1) to 4) in arbitrary described dna molecule:
1) nucleotides sequence is classified as the dna molecule of the 152-757 position of sequence 2 in sequence table；
2) nucleotides sequence is classified as the dna molecule of sequence 2 in sequence table；
3) under strict conditions with 1) or 2) the dna molecule hybridization that limits and encode albumen described in claim 1
The dna molecule of matter；
4) with 1) or 2) or 3) the dna molecule that limits has more than 90% homology and encode claim 1
The dna molecule of described protein.
4. the recombinant vector containing nucleic acid molecules described in Claims 2 or 3, expression cassette, transgenic cell line or weight
Group bacterium.
5. recombinant vector according to claim 4 it is characterised in that: described recombinant vector be recombinant expressed load
Body or recombinant cloning vector.
6. recombinant vector according to claim 5 it is characterised in that: in described recombinant expression carrier, start
The promoter of the transcription of described gene is 35s promoter.
7. the protein described in claim 1 or the nucleic acid molecules described in Claims 2 or 3 or claim 4-6
In arbitrary described recombinant vector, expression cassette, transgenic cell line or recombinant bacterium in following (a1) or (a2)
Application:
(a1) regulate and control plant stress tolerance；
(a2) plant variety that selection-breeding resistance of reverse improves.
8. a kind of method cultivating the transgenic plant that resistance of reverse improves, comprising:
A) import the encoding gene of protein described in claim 1 in recipient plant, obtain expressing described encoding gene
Transgenic plant；
B) obtain compared with described recipient plant from step a) gained transgenic plant, the transgenic that resistance of reverse improves
Plant.
9. according to claim 7 application or claim 8 described in method it is characterised in that: described coding
Gene is to import described recipient plant by described recombinant vector arbitrary in claim 4-6；And/or
Described resistance of reverse be following at least one: drought resistance, salt-resistance and aba toleration；And/or
Described plant is monocotyledon or dicotyledon.
10. the total length of encoding gene of protein or the primer pair of its arbitrary fragment described in amplification claim 1.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111675757A (en) * | 2020-07-16 | 2020-09-18 | 南京农业大学 | Du pear vacuole type proton pump PbVHA-B1 and application thereof in plant salt-resistant genetic improvement |
| CN111733277A (en) * | 2020-07-17 | 2020-10-02 | 中国农业科学院作物科学研究所 | Gene with salt-tolerant function and application thereof |
| CN113005126A (en) * | 2020-12-28 | 2021-06-22 | 四川农业大学 | DgSPL3 gene and cloning method and application thereof |
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|---|---|---|---|---|
| CN101955521A (en) * | 2010-09-21 | 2011-01-26 | 中国农业大学 | Plant stress tolerance associated protein, and coded genes and application thereof |
| CN103319583A (en) * | 2012-03-19 | 2013-09-25 | 中国农业科学院作物科学研究所 | Plant stress tolerance-associated protein TaNF-YB 1, coding genes thereof and applications |
| CN104744578A (en) * | 2015-02-03 | 2015-07-01 | 山东省农业科学院作物研究所 | Plant stress tolerance correlated protein as well as encoding gene ScMYB3R1 and applications thereof |
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2015
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101955521A (en) * | 2010-09-21 | 2011-01-26 | 中国农业大学 | Plant stress tolerance associated protein, and coded genes and application thereof |
| CN103319583A (en) * | 2012-03-19 | 2013-09-25 | 中国农业科学院作物科学研究所 | Plant stress tolerance-associated protein TaNF-YB 1, coding genes thereof and applications |
| CN104744578A (en) * | 2015-02-03 | 2015-07-01 | 山东省农业科学院作物研究所 | Plant stress tolerance correlated protein as well as encoding gene ScMYB3R1 and applications thereof |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111675757A (en) * | 2020-07-16 | 2020-09-18 | 南京农业大学 | Du pear vacuole type proton pump PbVHA-B1 and application thereof in plant salt-resistant genetic improvement |
| CN111675757B (en) * | 2020-07-16 | 2022-04-12 | 南京农业大学 | Du pear vacuole type proton pump PbVHA-B1 and application thereof in plant salt-resistant genetic improvement |
| CN111733277A (en) * | 2020-07-17 | 2020-10-02 | 中国农业科学院作物科学研究所 | Gene with salt-tolerant function and application thereof |
| CN111733277B (en) * | 2020-07-17 | 2021-04-13 | 中国农业科学院作物科学研究所 | Gene with salt-tolerant function and application thereof |
| CN113005126A (en) * | 2020-12-28 | 2021-06-22 | 四川农业大学 | DgSPL3 gene and cloning method and application thereof |
| CN113005126B (en) * | 2020-12-28 | 2022-06-24 | 四川农业大学 | DgSPL3 gene and cloning method and application thereof |
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