CN106337041B - Plant stress tolerance correlative protein and its encoding gene and application - Google Patents
Plant stress tolerance correlative protein and its encoding gene and application Download PDFInfo
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Abstract
The invention discloses a kind of plant stress tolerance correlative protein and its encoding gene and applications.Protein provided by the present invention, be following (a) or (b): (a) amino acid sequence is the protein of sequence 1 in sequence table；(b) amino acid sequence of protein defined by (a) is passed through to the substitution and/or deletion and/or addition of one or several amino acid residues, and protein relevant to plant stress tolerance.The present invention provides an economy, quickly and effectively approach to improve the resistance of reverse of plant；The present invention, by with wide application and market prospects, provides important candidate gene in agriculture field for genetic engineering Crop Improvement resistance of reverse in future.
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 technique
The abiotic stress such as arid, saline and alkaline, extreme temperature cause the growth of crop and seriously threaten, and generate to plant
A series of morphology, the influence of Physiology and biochemistry state and molecular level cause vine growth and development slow, even draw when serious
Irreversible injury is played, entire plant is even resulted in and shifts to an earlier date death, greatly influence the yield of crop.Worldwide, non-life
Object adverse circumstance is to occupy the 50% of crop failure every year on average the main reason for causing crop failure.Under the conditions of environment stress, plant
The object biosystem complicated as one, often generates a series of forms, Physiology and biochemistry, molecule, gene and metabolite etc.
Horizontal variation, to generate certain resistance or patience to adverse circumstance.
Since plant is perphyton, adverse circumstance cannot be hidden by shift position when in face of poor environment.In order to survive,
Plant evolution goes out a series of raising adversity stress resistances or patience strategy.Research in relation to plant stress-resistance, resistance of reverse is always to plant
The research hotspot in object field.Resistance, the resistance of reverse difficulty for improveing plant by traditional breeding technique are relatively large, cannot
More excellent degeneration-resistant, resistance to inverse kinds are obtained well.And with the development of molecular biology technology, and to plant stress-resistance,
The further investigation of resistance to inverse molecular mechanism, degeneration-resistant (resistance to inverse) genetic engineering of molecular level make substantial progress.Currently, using transgenosis
Etc. genetic engineering means to plant import external source resistance of reverse gene have become improvement plant stress tolerance one of new way.Research is planted
Object is degeneration-resistant, resistance to inverse molecular regulation network and turns out more excellent drought-enduring varieties and has boundless prospect and particularly significant
Meaning.
Under the conditions of environment stress, plant can be coerced adverse circumstance by a series of physiology, Biochemical changes and metamorphosis
Compel to generate certain adaptation.Vacuole is the coated balloon-shaped structure of the distinctive film of plant cell, accounts for cell body in mature cell
Long-pending 90% or more.Under the conditions of environment stress, plant adjusts Premeabilisation of cells pressure by vacuole, maintains intracellular water flat
Weighing apparatus, accumulation and storage nutrient, a variety of metabolites and noxious material, reach material energy metabolism, life under the conditions of environment stress
Long and development balance.V-ATPase (vacuole v-shaped atp synthase) is the important enzyme on vacuole, is turned with the substance across tonoplast
Fortune has substantial connection, and V-ATPase generates the electrochemical gradient of cross-film simultaneously by the energy transfer proton that hydrolysising ATP generates
The pH value of intraor extracellular is adjusted, the Na in cytoplasm is promoted+Etc. being transferred in vacuole and accumulate, Na is eliminated+Murder by poisoning to cell.
Therefore, V-ATPase has extremely important effect during the degeneration-resistant Stress responses of plant.V-ATPase is by multiple subunits
" bulb " structure is presented in composition, space structure.Part outside prominent film is known as V1, and film inner part is known as V0.The part V1 about 8
Kind subunit composition is respectively designated as A~H according to molecular size range, and the part V0 is about made of three kinds of subunits.Wherein V1 part
E subunit is a hydrophilic protein with abundant charged residues, has crucial make in the assembling process of V-ATPase albumen
With.
Summary of the invention
The object of the present invention is to provide a kind of plant stress tolerance correlative protein and its encoding gene and applications.
Protein provided by the present invention, entitled sorghum vacuole atp synthase E protein subunit (SbATPase-E), source
It is following (a) or (b) in sorghum (Sorghum bicolor L.):
(a) amino acid sequence is the protein of sequence 1 in sequence table；
(b) by the amino acid sequence of protein defined by (a) by one or several amino acid residues substitution and/or
Deletion and/or addition, and protein relevant to plant stress tolerance.
It, can the amino acid residue sequence of sequence 1 forms in by sequence table egg for the ease of the purifying of the protein
The upper label as shown in the table of amino terminal or carboxyl terminal connection of white matter.
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 be artificial synthesized, can also first synthesize its encoding gene, then carries out biological expression and obtain.
The encoding gene of protein in above-mentioned (b) can be one or several by will lack in DNA sequence dna shown in sequence 2 in sequence table
The codon of amino acid residue, and/or carry out the missense mutation of one or several base-pairs.
The nucleic acid molecules of code for said proteins also belong to protection scope of the present invention.
The nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules are also possible to
RNA, such as mRNA, hnRNA or tRNA.
In one embodiment of the invention, the nucleic acid molecules are specially the gene of code for said proteins；The base
Because can be to be following 1) to the DNA molecular any in 4):
1) nucleotides sequence is classified as 152-757 DNA moleculars of sequence 2 in sequence table；
2) nucleotides sequence is classified as the DNA molecular of sequence 2 in sequence table；
1) or 2) 3) hybridize under strict conditions with the DNA molecular limited and the DNA molecular of code for said proteins；
1) or 2) or 3) 4) there are the DNA of 90% or more homology and code for said proteins points with the DNA molecular limited
Son.
Above-mentioned stringent condition can for 6 × SSC, the solution of 0.5%SDS hybridizes at 65 DEG C, then with 2 × SSC,
It is primary that 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film.
Wherein, sequence 2 is made of 1016 nucleotide, and 152-757 are ORF, in polynucleotide shown in sequence 1
Protein.
Recombinant vector, expression cassette, transgenic cell line or recombinant bacterium containing above-mentioned nucleic acid molecules also belong to of the invention
Protection scope.
The recombinant vector can be recombinant expression carrier, can also be recombinant cloning vector.
The recombinant expression carrier can use existing plant expression vector construction.The plant expression vector includes double base agriculture
Bacillus carrier and the carrier etc. that can be used for plant micropellet bombardment, as pGreen0029, pCAMBIA3301, pCAMBIA1300,
The derivative plant expression vector of pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other.The plant expression
Carrier also may include 3 ' end untranslated regions of foreign gene, i.e., processes comprising polyadenylation signals and any other participation mRNA
Or the DNA fragmentation of gene expression.The bootable polyadenylic acid of polyadenylation signals is added to 3 ' ends of mRNA precursor.Using institute
It, can be plus any enhanced, composing type, tissue before its transcription initiation nucleotide when stating gene constructed recombinant expression carrier
Idiotype or inducible promoter, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin starting
Sub (pUbi), stress induced promoter rd29A etc., they can be used alone or are used in combination with other plant promoters；
In addition, also enhancer, including translational enhancer or transcription can be used to increase when using gene constructed recombinant expression carrier of the invention
Hadron, these enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but required and coded sequence
Reading frame it is identical, to guarantee the correct translation of entire sequence.The source of the translation control signal and initiation codon is wide
It is general, it can be natural, be also possible to synthesis.Translation initiation region can come from transcription initiation region or structural gene.
For the ease of transgenic plant cells or plant are identified and screened, recombinant expression carrier used can be processed, such as
The coding that being added can express in plant can produce the enzyme of color change or the gene of luminophor, resistant antibiotic
Marker or anti-chemical reagent marker gene etc..Any selected marker can also be not added, directly screened and converted with adverse circumstance
Plant.
In an embodiment of the present invention, the promoter for starting the genetic transcription in the recombinant expression carrier is specially
35S promoter.
More specifically, the recombinant expression carrier is the restriction enzyme site BglII and BstE II in pCAMBIA3301 carrier
Between the recombinant plasmid that is obtained after DNA fragmentation shown in sequence 3 in insetion sequence table.
The expression cassette is by that can start the promoter of the gene expression, the gene and transcription terminator group
At.
The transgenic cell line is non-propagation material.
The protein or the nucleic acid molecules or the recombinant vector, expression cassette, transgenic cell line or recombinant bacterium exist
The application in (a1) or (a2) also belongs to protection scope of the present invention as follows:
(a1) regulate and control plant stress tolerance；
(a2) plant variety that breeding resistance of reverse improves.
In the present invention, the regulation plant stress tolerance is embodied in: in the plant, if the expression of the protein
In a certain range, expression quantity is higher, then the resistance of reverse of the plant is stronger for amount；If the expression quantity of the protein is lower,
The resistance of reverse of the plant is weaker.
In the present invention, the method for the plant variety that the breeding resistance of reverse improves, specifically may include by the protein
The step of higher plant of expression quantity hybridizes as parent.
It is a further object to provide a kind of methods of genetically modified plants that cultivation resistance of reverse improves.
The method provided by the present invention for cultivating the genetically modified plants that resistance of reverse improves, comprising:
A) encoding gene that the protein is imported into recipient plant, the transgenosis for obtaining expressing the encoding gene are planted
Object；
B) it is obtained compared with the recipient plant from genetically modified plants obtained by step a), the transgenosis that resistance of reverse improves is planted
Object.
The gene can be to be following 1) to the DNA molecular any in 4):
1) nucleotides sequence is classified as 152-757 DNA moleculars of sequence 2 in sequence table；
2) nucleotides sequence is classified as the DNA molecular of sequence 2 in sequence table；
1) or 2) 3) hybridize under strict conditions with the DNA molecular limited and the DNA molecular of code for said proteins；
1) or 2) or 3) 4) there are the DNA of 90% or more homology and code for said proteins points with the DNA molecular limited
Son.
Above-mentioned stringent condition can for 6 × SSC, the solution of 0.5%SDS hybridizes at 65 DEG C, then with 2 × SSC,
It is primary that 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film.
The encoding gene can specifically be imported in the purpose plant by any of the above-described recombinant expression carrier, be obtained
The genetically modified plants.Specifically can by using Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection,
The recombinant expression carrier is converted plant cell or tissue by the conventional biology methods such as conductance, mediated by agriculture bacillus, particle gun, and
The plant tissue of conversion is cultivated into plant.
In above-mentioned application or method, the resistance of reverse is concretely at least one of following: drought resistance, salt-resistance
With ABA tolerance.The resistance of reverse is presented as the resistance of reverse in Their Seed Germinating Period and/or seedling stage.
The plant is dicotyledon or monocotyledon.The dicotyledon is crucifer, such as quasi- south
Mustard.The monocotyledon is gramineae plant, such as sorghum.
The primer pair of the overall length or its any segment that expand the encoding gene of the protein also belongs to protection of the invention
Range.
The present invention provides an economy, quickly and effectively approach to improve the resistance of reverse of plant；The present invention is led in agricultural
Domain provides important candidate base for wide application and market prospects for genetic engineering Crop Improvement resistance of reverse in future
Cause.
Detailed description of the invention
Fig. 1 is the building flow chart of recombinant expression carrier pCAMBIA3301-35S::SbATPase-E.
The bacterium solution PCR that Fig. 2 is recombinational agrobacterium AGl0/pCAMBIA3301-35S::SbATPase-E identifies electrophoretogram.Its
In, swimming lane M is DNA molecular amount standard；Remaining swimming lane is positive restructuring Agrobacterium AGl0/pCAMBIA3301-35S::
SbATPase-E。
Fig. 3 is that T3 is identified for the PCR of transgenic arabidopsis homozygous lines, and PCR the primer is vector primer, target fragment
Overall length 1500bp or so.Wherein, swimming lane M is DNA molecular amount standard；Swimming lane 1 and 2 is respectively positive T3 pure for transgenic arabidopsis
Close strain 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 processing of 175mM.
Fig. 5 is that sprouting stage wildtype Arabidopsis thaliana and T3 fall off for transgenic line FOX30.3 and FOX30.4 at 0.2 μm
Phenotype under acid processing.
Fig. 6 be wildtype Arabidopsis thaliana and T3 for transgenic line FOX30.3 the NaCl and 250mM of 150mM mannitol
Phenotype after handling 1 week.
Specific embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
The drought-enduring sorghum variety XGL-1 in Xinjiang: Grain Crops Inst., Xinjiang Agricultural Academy.It is recorded in " Lu Min corn
The clone of ZmSNAC1 and sorghum SbSNAC1 gene and the functional analysis Chinese Academy of Agricultural Sciences, crop germplasm resource, 2013,
A doctoral thesis " text, the public can obtain from applicant, can only be used to repeat present invention experiment.
PDONR222 carrier: Invitrogen Products, while being also recorded in " COPT6is a Plasma
Membrane Transporter that Functions in Copper Homeostasis in Arabidopsis and
7 " one text of is a Novel Target of SQUAMOSA Promoter Binding Protein-Like, the public can be from Shen
It asks someone to locate to obtain, can only be used to repeat present invention experiment.
PMD18-T carrier: Takara, Catalog no.D101A.
PCAMBIA3301 plasmid: it is recorded in " A maize stress-responsive NAC transcription
factor,ZmSNAC1,confers enhanced tolerance to dehydration in transgenic
Arabidopsis, Plant Cell Rep, 2012 " one texts, the public can obtain from applicant, can only be used to repeat the present invention
Experiment.
Agrobacterium AGl0: be recorded in " mediated by agriculture bacillus gna genetic transformation Elite Maize Inbred Lines and transgenic corns it is anti-
Worm Disease-resistance Analysis, Shandong University Ph.D. Dissertation in a 2005 " text, the public can obtain from applicant, can only be used to weight
Duplicate invention experiment.
Arabidopsis (Columbia-0): it is recorded in " A maize stress-responsive NAC transcription
factor,ZmSNAC1,confers enhanced tolerance to dehydration in transgenic
Arabidopsis, Plant Cell Rep, 2012 " one texts, the public can obtain from applicant, can only be used to repeat the present invention
Experiment.
The discovery of embodiment 1, SbATPase-E protein subunit and its encoding gene
It is building up on expression vector by sorghum cDNA library, extensive arabidopsis thaliana transformation, by a large amount of drought resistances
Shape identification and Molecular Detection find a new albumen from the drought-enduring sorghum variety XGL-1 in Xinjiang, are named as SbATPase-E subunit
Albumen.
The amino acid sequence of SbATPase-E protein subunit is sequence 1 in sequence table.By SbATPase-E protein subunit
Encoding gene is named as SbATPase-E gene, and cDNA is as shown in the sequence 2 of sequence table.
The blade total serum IgE of the drought-enduring sorghum variety XGL-1 in Xinjiang of Osmotic treatment is extracted, and reverse transcription is cDNA.With cDNA
As template, PCR amplification is carried out using primers F and R, sequencing is carried out to gained PCR product.PCR product as the result is shown
Sequence is just being in sequence table shown in sequence 2.
Primers F: 5 '-ACAAGTTTGTACAAAAAAGTT-3 '；
Primer R:5 '-GATTACACCAAGTGCGGGTGT-3 '.
Embodiment 2, the acquisition of transgenic plant and resistance of reverse identification
One, the acquisition of transgenic plant
1, the building of recombinant expression carrier
The building flow chart of recombinant expression carrier is shown in Fig. 1.
(1) total serum IgE of the drought-enduring sorghum variety XGL-1 in Xinjiang of Osmotic treatment is extracted, and reverse transcription is cDNA.Made with cDNA
For template, PCR amplification is carried out using primers F and R (sequence is same as above) and obtains SbATPase-E gene shown in sequence 2 in sequence table
SbATPase-E gene (sequence 2) is recombinated by Gateway (BP) reaction and is imported by (can certainly artificial synthesized sequence 2)
Gained recombinant plasmid is named as pDONR222-SbATPase-E by pDONR222 carrier.
(2) the identification sequence for adding restriction enzyme site Bgl II and BstE II respectively at the both ends Gateway Sequence, obtains
DNA fragmentation shown in sequence 4 (is followed successively by restriction enzyme site Bgl II, attR1, ccdB, attR2 and restriction enzyme site from 5 ' ends to 3 ' ends
BstE II), and obtain carrying out Bgl II and II digestion of BstE after recombinant plasmid after pMD18-T carrier, recycling and purifying digestion products
(purpose band that size is about 2430bp).
(3) II double digestion pCAMBIA3301 plasmid of restriction enzyme BglII and BstE is used, recycling carrier framework is (about
9250bp), the GUS segment between BglII and BstE II is cut off.
(4) the digestion recovery product of step (3) is connected with the carrier endonuclease bamhi of step (2), obtains recombinant plasmid
pCAMBIA3301-attR1-ccdB-attR2.The structure of the recombinant plasmid pCAMBIA3301-attR1-ccdB-attR2 is retouched
It states are as follows: replace with the small fragment (gus gene) in pCAMBIA3301 plasmid between restriction enzyme site BglII and BstE II
The recombinant plasmid obtained after DNA fragmentation shown in sequence 4 in sequence table.
(5) being reacted by LR will be in recombinant plasmid pDONR222-SbATPase-E obtained in step (1) shown in sequence 2
SbATPase-E gene and step (4) in obtain the ccdB base in recombinant plasmid pCAMBIA3301-attR1-ccdB-attR2
Because recombination exchange occurs, recombinant plasmid pCAMBIA3301-35S::SbATPase-E is obtained.The recombinant plasmid
PCAMBIA3301-35S::SbATPase-E structure is described as follows: will be located at restriction enzyme site BglII in pCAMBIA3301 plasmid
Small fragment (gus gene) between BstE II replaces with the recombinant plasmid obtained after DNA fragmentation shown in sequence 3 in sequence table.
2, the acquisition and identification of genetically modified plants
1) step 1 is constructed into resulting recombinant expression carrier pCAMBIA3301-35S::SbATPase-E and converts Agrobacterium
AGl0 obtains recombinational agrobacterium.To recombinational agrobacterium, bacterium solution PCR identification is carried out using primers F 1 and R1.Positive Agrobacterium
PCR is identified as shown in Fig. 2, obtaining the purpose band that size is about 695bp.
Primers F 1:5 '-ATGAATGACACCGATGTAGCCAA-3 ' (152-173 of sequence 2)；
Primer R1:5 '-TTAGGCTGCAGTCTGGCCA-3 ' (828-846 reverse complementary sequences of sequence 2).
The plasmid for extracting the positive recombinational agrobacterium of PCR identification sends to sequencing, and extracted plasmid is just as the result is shown
PCAMBIA3301-35S::SbATPase-E (sequencing show will in pCAMBIA3301 plasmid be located at restriction enzyme site BglII and
Small fragment (gus gene) between BstE II replace with DNA fragmentation shown in sequence 3 in sequence table (sequence 3 from 5 ' end to 3 ' end according to
Secondary is attB1, SbATPase-E, attB2) after obtained recombinant plasmid), illustrate to be named as positive restructuring Agrobacterium
AGl0/pCAMBIA3301-35S::SbATPase-E。
2) recombinational agrobacterium AGl0/pCAMBIA3301-35S::SbATPase-E is cultivated, 28 DEG C (200rmp) shakes
Swinging to OD600 value is 1.2-1.4.Thallus is collected, (is formulated with suitable permeabilization buffer: being added 5% in 1/2MS culture medium
(5g/100ml) sucrose) sufficiently suspension thalline to bacterium solution OD600 value be 0.8 or so, to the bacterium solution after being suspended with permeabilization buffer
In addition final concentration of 0.02% (volume fraction) adsorbent Silwet L-77, obtain Agrobacterium bacterium solution.
3) ready arabidopsis (Columbia-0) petal is completely soaked in the resulting Agrobacterium bacterium solution of step 2)
About 1min is infected, inflorescence is gently shaken and inflorescence is sufficiently immersed in bacterium solution, plant is transferred to by 16 DEG C of dark processings afterwards for 24 hours
Growth repeats conversion 1-2 times under normal growing conditions, mixes and receives T0 for arabidopsis seed.
4) T0 is laid in nutritive cube for arabidopsis seed, each nutritive cube about spreads 1000 seeds, opens after emergence
Begin to carry out Osmotic treatment, when Arabidopsis plants most in nutritive cube are dead, the plant to survive in each nutritive cube only has 1-5
Rehydration processing is carried out when strain, then single plant harvests seed, obtains T1 for transgenic line.
To T1 for drought-enduring positive transgenic strain single plant sowing, on the MS plate containing cremart (PPT) (7mg/L)
It identifies (due to carrying cremart resistant gene Bar on pCAMBIA3301 carrier), and screens the transgenosis of T2, T3 generation homozygosis
Strain (segregation ratio identification), parent's strain that all offsprings all have cremart resistance is homozygous lines.Use with
The primers F 2 and R2 of pCAMBIA3301 vector backbone sequence design carry out the homozygous lines obtained by cremart Resistance Identification
Molecular Identification.Simultaneously using the arabidopsis (Columbia-0) of non-transgenosis as wild type control (WT).
Primers F 2:5 '-TCTCCACTGACGTAAGGGAT-3 ' (2579-2598 of sequence 3)；
Primer R2:5 '-TTAGGTTTACCCGCCAATAT-3 ' (4019-4038 reverse complemental sequences of sequence 3
Column).
Wherein, the PCR qualification result of T3 generation positive arabidopsis homozygous lines is as shown in figure 3, obtaining expected size and being about
The purpose band of 1460bp.And wild type control does not obtain purpose band through expanding.The T3 positive from identification is for the quasi- south of transgenosis
Two strains are randomly selected in mustard homozygous lines, are denoted as FOX30.3 and FOX30.4 respectively, are identified for subsequent resistance of reverse.
It tests while unloaded control is set, it may be assumed that be transferred to pCAMBIA3301- into Agrobacterium AGl0 referring to method as above
(pCAMBIA3301-35S:: Δ GUS carrier is with II double digestion of restriction enzyme BglII and BstE to 35S:: Δ GUS carrier
Gained is connected after the skeleton carrier end-filling for being about 9250bp by resulting size after pCAMBIA3301 plasmid), by gained weight
Group Agrobacterium is named as AGl0/pCAMBIA3301-35S:: Δ GUS.With further reference to method as above by recombinational agrobacterium AGl0/
PCAMBIA3301-35S:: Δ GUS imports arabidopsis (Columbia-0), obtains T0 for arabidopsis seed.By selfing and such as
Upper cremart resistance screening obtains T3 for homozygous lines.Also pass through the PCR amplification of primers F 2 and R2 as above, gained PCR product
Size be about 444bp, it is consistent with expected results.
Two, the resistance of reverse identification of transgenic plant
1, germination period stress experiment
By the T3 screened through step 1 for the seed of homozygous transgenic arabidopsis strain FOX30.3 and FOX30.4, with
And it is transferred to the seed of the T3 generation homozygous arabidopsis strain of empty carrier and the kind of wildtype Arabidopsis thaliana (Columbia-0) (WT) strain
Son carries out disinfection, respectively by its program request in the MS culture medium for containing various concentration NaCl (150mM, 175mM) and ABA (0.2 μM)
It is handled on control MS culture medium, 5 repetitions are arranged in each 35 seeds of strain point, and the seed on plate is protected from light
It is placed in 4 DEG C vernalization 2-4 days, the seed by vernalization, which is put between culture, sprouts (22 DEG C of illumination/18 16h under normal growing conditions
DEG C 8h dark, relative air humidity 40%-50%), the seed germination rate of each processing is counted after 7 days.
As the result is shown:
(1) tolerance of NaCl is analyzed
On control MS culture medium, two separate transgenic strains of FOX30.3 and FOX30.4 and wild type control strain
(WT) germination rate between is without significant difference.And on the MS plate containing 150mM, 175mM NaCl, FOX30.3 and FOX30.4
Transgenic line shows apparent salt tolerance compared with WT strain (WT).Under 150mM NaCl existence condition, about 33%
Wildtype Arabidopsis thaliana (WT) seed is sprouted, and the germination rate of FOX30.3 and FOX30.4 this 2 transgenic lines respectively reaches
72% and 56%, it is significantly higher than the germination rate (P < 0.05) of wild type (WT) arabidopsis.Under 175mM NaCl existence condition, about
2.7% wildtype Arabidopsis thaliana (WT) seed is sprouted, and the germination rate difference of FOX30.3 and FOX30.4 this 2 transgenic lines
Reach 25.7% and 20.0%, is significantly higher than the germination rate (P < 0.05) of wildtype Arabidopsis thaliana (WT) seed.As it can be seen that seed is sprouted
Stage, the overexpression of SbATPase-E gene significantly improve transgenic line to the resistance (such as Fig. 4) of salt stress.
Either on control MS culture medium, or on the MS plate containing 150mM or 175mM NaCl, it is transferred to zero load
The germination rate of the seed of the T3 generation homozygous arabidopsis strain of body is almost the same with wildtype Arabidopsis thaliana (WT), and no statistics is poor
It is different.
(2) tolerance of ABA is analyzed
The sprouting of two separate transgenic strains of FOX30.3 and FOX30.4 and wildtype Arabidopsis thaliana (WT) under ABA stress
Rate is inhibited to a certain extent.Under the conditions of existing for 0.2 μM of ABA, FOX30.3 and FOX30.4 this 2 turn base
Because strain has the seed of 70%-76% to sprout, and the germination rate of wild type (WT) seed only has 28%, the two significant difference (P <
0.05) (Fig. 5).Result of study shows that ABA is substantially less than WT lines (WT) to the inhibiting effect that transgenic line is sprouted.
On the MS plate containing 0.2 μM of ABA, it is transferred to the sprouting of the seed of the T3 generation homozygous arabidopsis strain of empty carrier
Rate and wildtype Arabidopsis thaliana (WT) are almost the same, no difference of science of statistics.
2, seedling stage stress experiment
By 4 -day-old of the T3 screened through step 1 for homozygous transgenic arabidopsis strain FOX30.3 and FOX30.4,
And it is transferred to the seedling point of T3 the generation homozygous arabidopsis strain and wildtype Arabidopsis thaliana (Columbia-0) strain (WT) of empty carrier
It is not transferred to normal MS culture medium, contains 150mmolL-1NaCl or 350mmolL-1On the MS plating medium of mannitol,
Plate is disposed vertically, and growth of seedling counts the leaf blade size, root length, lateral root number isophenous of each group seedling after a week
Shape.Each each 12 seedling of repetition of strain, are arranged 3 repetitions.
As the result is shown:
(1) (salt-resistance) is analyzed to the tolerance of NaCl
One week wild type (WT) is grown on normal MS culture medium and FOX30.3 and FOX30.4 transgenic line ties up to leaf
Piece size, root length do not have notable difference in lateral root number isophenous character.Containing 150mmolL-1The MS of NaCl is trained
The growth for supporting FOX30.3 the and FOX30.4 transgenic line and WT strain that grow on base is all significantly inhibited, still
NaCl is affected to WT lines, blade obviously become smaller compared with FOX30.3 and FOX30.4 transgenic line (P <
0.05), root system obviously shortens compared with FOX30.3 and FOX30.4 transgenic line, and wherein transgenic line is 4.2cm, wild type strain
The only 3.4cm of system, significant difference (P < 0.05) (Fig. 6).
Either on normal MS culture medium, or containing 150mmolL-1On the MS plate of NaCl, it is transferred to empty carrier
T3 generation homozygous arabidopsis strain plant phenotype it is almost the same with wildtype Arabidopsis thaliana, no difference of science of statistics.
(2) (drought resistance) is analyzed to the tolerance of mannitol
Containing 350mmolL-1On the MS culture medium of mannitol, FOX30.3 and FOX30.4 transgenic line and wild
The growth of type strain (WT) is also all significantly inhibited, but FOX30.3 and FOX30.4 transgenic plant is compared with WT lines
(WT) more healthy and stronger, transgenic plant root system is more flourishing, and transgenic line tip of a root number is dramatically increased compared with wild type, transgenosis
The strain tip of a root number that is averaged is 4.8, and wild type be averaged tip of a root number as 3.5 (P < 0.05).In addition, transgenic plant aerial part is still
Green so is shown as, and WT lines (WT) aerial part has occurred and that browning (Fig. 6).
Containing 350mmolL-1On the MS plate of mannitol, it is transferred to the T3 generation homozygous arabidopsis strain plant of empty carrier
Phenotype and wildtype Arabidopsis thaliana (WT) it is almost the same, no difference of science of statistics.
In summary, it is seen that SbATPase-E gene provided by the present invention helps to improve the salt tolerance, drought-enduring of plant
Property and tolerance to ABA.
Claims (11)
1. protein is the protein that amino acid sequence is sequence 1 in sequence table.
2. encoding the nucleic acid molecules of protein described in claim 1.
3. nucleic acid molecules according to claim 2, it is characterised in that: the nucleic acid molecules are described in coding claim 1
The gene of protein；The gene be it is following 1) or 2) described in DNA molecular:
1) nucleotides sequence is classified as 152-757 DNA moleculars of sequence 2 in sequence table；
2) nucleotides sequence is classified as the DNA molecular of sequence 2 in sequence table.
4. the recombinant vector containing nucleic acid molecules described in Claims 2 or 3.
5. the expression cassette containing nucleic acid molecules described in Claims 2 or 3.
6. the recombinant bacterium containing nucleic acid molecules described in Claims 2 or 3.
7. recombinant vector according to claim 4, it is characterised in that: the recombinant vector is recombinant expression carrier or recombination
Cloning vector.
8. recombinant vector according to claim 7, it is characterised in that: in the recombinant expression carrier, promotor gene transcription
Promoter be 35S promoter.
9. described in protein described in claim 1 or nucleic acid molecules described in claim 2 or 3 or claim 4 or 7 or 8
Recombinant vector or claim 5 described in expression cassette or recombinant bacterium as claimed in claim 6 in following (a1) or (a2)
Using:
(a1) regulate and control plant stress tolerance；
(a2) plant variety that breeding resistance of reverse improves；
The resistance of reverse is at least one of following: drought resistance, salt-resistance and ABA tolerance；
The plant is arabidopsis.
10. a kind of method for cultivating the genetically modified plants that resistance of reverse improves, comprising:
A) encoding gene that protein described in claim 1 is imported into recipient plant obtains expressing turning for the encoding gene
Gene plant；
B) it is obtained compared with the recipient plant from genetically modified plants obtained by step a), the genetically modified plants that resistance of reverse improves；
The resistance of reverse is at least one of following: drought resistance, salt-resistance and ABA tolerance；
The plant is arabidopsis.
11. according to the method described in claim 10, it is characterized by: the encoding gene is by claim 4 or 7 or 8
The recombinant vector imports the recipient plant.
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| 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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| 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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