CN106854238B - Plant adversity resistance related protein TabZIP14 and its encoding gene and application - Google Patents
Plant adversity resistance related protein TabZIP14 and its encoding gene and application Download PDFInfo
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Abstract
The invention discloses a plant resistance relevant protein TabZIP14 and its encoding gene and applications.TabZIP14 protein provided by the present invention, specifically following (a) or (b): (a) protein that the amino acid sequence shown in sequence 1 in sequence table forms；(b) amino acid sequence of sequence 1 is passed through to the substitution and/or deletion and/or addition of one or several amino acid residues, and the protein as derived from sequence 1 relevant to stress resistance of plant.TaAREB3 albumen and its encoding gene provided by the present invention are of great significance in terms of improving stress resistance of plant, will play a significant role in cultivating high resistance to cold and diseases such as strong frost resistance and strong salt-resistance plant variety.
Description
Technical field
The invention belongs to plant genetic engineering fields, are related to a plant resistance relevant protein TabZIP14 and its coding
Gene and application.
Background technique
Abiotic stress is to influence one of crop growth and the principal element of yield, develops and utilizes anti-adversity and exists
Salt-soda soil and water-deficient area are particularly important.Some progress are obtained in terms of the gene for improving stress resistance of plant recent years.
Transcription factor is also in response to the key regulator of plant abiotic stress adverse circumstance, and being overexpressed some transcription factor genes can
To improve the resistance of plant.BZIP is exactly to be present in one of plant transcription factor.
A series of responsing reactions can be generated in plant under environment stress, along with many Physiology and biochemistries and developmentally
Variation.Plant is specified to the reaction mechanism of adverse circumstance, scientific argument will be provided for adversity gene engineering research and application.Currently, planting
The degeneration-resistant Journal of Sex Research of object is gradually deep into cell, molecular level, and combines with science of heredity and genetic engineering research, explores with life
Object technology improves plant growth characteristics, and the purpose is to improve plant to the adaptability of adverse circumstance.
Therefore, using Modern Molecular Biotechnology, degeneration-resistant key gene is excavated, plant is improved by genetic engineering and is especially
The resistance of crops, for ensureing that national food security is of great significance.
Summary of the invention
The object of the present invention is to provide a plant resistance relevant protein TabZIP14 and its encoding gene and applications.
TabZIP14 albumen source provided by the present invention in wheat (Triticum aestivum L.) kind China spring,
Concretely following (a) or (b):
(a) protein that the amino acid sequence shown in sequence 1 in sequence table forms；
(b) amino acid sequence of sequence 1 by the substitution of one or several amino acid residues and/or missing and/or is added
Add, and the protein as derived from sequence 1 relevant to stress resistance of plant.
For the ease of the purifying of protein shown in above-mentioned (a), can in by sequence table sequence 1 amino acid residue sequence
The upper label as shown in the table of amino terminal or carboxyl terminal connection of the protein of composition.
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 of concretely following 1) -4) in any DNA molecular:
1) DNA molecular shown in sequence 2 in sequence table；
2) DNA molecular shown in 181-1383 of sequence 2 in sequence table；
1) or 2) 3) hybridize under strict conditions with the DNA molecular limited and encode the DNA molecular of anti reversion relative protein；
4) with 1) -3) in the DNA sequence dna of any restriction there is 90% or more identity, and encode anti reversion relative protein
DNA molecular.
Wherein, sequence 2 is made of 1576 nucleotide, and 181-1383 are ORF, in polynucleotide shown in sequence 1
Protein.
Recombinant vector, expression cassette, transgenic cell line or recombinant microorganism containing above-mentioned nucleic acid molecules also belong to this hair
Bright 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 the present invention, the promoter for starting the genetic transcription in the recombinant expression carrier is specially 35S promoter.
Further, the recombinant expression carrier be will be small between the attR1 and attR2 of 100 carrier of pEarleyGate
Segment replaces with the recombinant plasmid obtained after the gene.
More specifically, the recombinant expression carrier is anti-by BP by the gene (181-1383 of sequence 2)
It should recombinate on entry vector, obtain introduction plasmid；The purpose weight that plasmid of geting started is reacted with destination carrier by LR again
Group plasmid；The entry vector is specially that (carrier is specially invitrogen Products, catalog number to pDONR/Zeo
For 12535-035)；The destination carrier is specially that (carrier is specially to produce from invitrogen company to pEarleyGate 100
Product)；The pEarleyGate 100 is in document " Zhang L, Zhang L, Xia C, Zhao G, Liu J, Jia J, Kong
X.A novel wheat bZIP transcription factor,TabZIP60,confers multiple abiotic
stress tolerances in transgenic Arabidopsis.Physiol Plant.2015,153(4):538-
It is disclosed in 554. ".
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 the non-propagation material for being transferred to the gene.
The protein or the nucleic acid molecules or the recombinant vector, expression cassette, transgenic cell line or the micro- life of recombination
Object it is following it is any in application also belong to protection scope of the present invention:
(a) regulate and control stress resistance of plant；
(b) plant variety that breeding resistance improves.
In (a), the resistance of the regulation plant is embodied in: in the plant, if the gene or institute
The expression quantity for stating albumen is higher, then the resistance of the plant is stronger；If the expression quantity of the gene or the albumen is lower,
The resistance of the plant is weaker.
In (b), the method for the plant variety of breeding resistance enhancing specifically may include by the gene or described
The step of higher plant of expressing quantity hybridizes as parent.
The present invention also provides a kind of methods of genetically modified plants that cultivation resistance improves.
The method provided by the present invention for cultivating the genetically modified plants that resistance improves, specifically may include following (a1) and
(a2) the step of:
(a1) encoding gene that the protein is imported into recipient plant obtains the transgenosis for expressing the encoding gene
Plant；
(a2) it is obtained compared with the recipient plant from genetically modified plants obtained by step (a1), what resistance improved turns base
Because of plant.
Expression quantity of the protein in the genetically modified plants is higher than the recipient plant；Code for said proteins
Gene is following 1) -4) in any DNA molecular:
1) DNA molecular shown in sequence 2 in sequence table；
2) DNA molecular shown in 181-1383 of sequence 2 in sequence table；
1) or 2) 3) hybridize under strict conditions with the DNA molecular limited and encode the DNA molecular of anti reversion relative protein；
4) with 1) -3) in the DNA sequence dna of any restriction there is 90% or more identity, and encode anti reversion relative protein
DNA molecular.
The gene can specifically be imported in the recipient plant by any of the above-described recombinant expression carrier, be obtained described
Genetically modified plants.It specifically can be by using Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity
Lead, the recombinant expression carrier is converted plant cell or tissue by the conventional biology methods such as mediated by agriculture bacillus, particle gun, and will
The plant tissue of conversion is cultivated into plant.
In the present invention, the gene is imported by recombinational agrobacterium, and the recombinational agrobacterium is by the recombination
Expression vector imports what Agrobacterium obtained；The Agrobacterium is specially Agrobacterium GV3101-pMP 90, the bacterium document " Xu Z,
Kim S,Hyeon D,Kim D,et al.The Arabidopsis thaliana NAC transcription factor
family:structure–function relationships and determinants of ANAC019 stress
Signaling.Biochem.J.2010,426:183-196. it is disclosed in ".
In above-mentioned application or method, the resistance is at least one of following: frost resistance, salt-resistance.
In the present invention, " jelly " in the frost resistance is embodied as -10 DEG C of processing 3h.
In the present invention, " salt " in the salt-resistance is embodied as 150-200mM NaCl processing.
In above-mentioned application or method, it can also be monocotyledon that the plant, which can be dicotyledon,.Such as: wheat, cigarette
Grass or arabidopsis etc..
In the present invention, the plant is arabidopsis, specially arabidopsis Columbia-0 hypotype.
It is demonstrated experimentally that by the recombinant expression carrier of DNA molecular shown in sequence 2 in expressible nucleotide sequence table (TabZIP14 gene)
The transgenic plant that arabidopsis thaliana transformation obtains, compared with the WT lines under the same terms: in frost resistance experiment (at -10 DEG C
Manage 3h) in, the survival rate of WT lines is 16%, and the survival rate for turning the strain of TabZIP14 gene is 79%.In salt resistance reality
In testing, after 150mM NaCl processing, the root long of wildtype Arabidopsis thaliana is obviously suppressed, and T3 generation turns TabZIP14 gene arabidopsis
The root long of L1, L2 and L3 strain is 1.4 times, 1.3 times and 1.4 times of WT lines root long, the quasi- south of Part Wild type respectively
Albinism is presented in mustard leaf, and albefaction rate is 37% ± 0.5%, and T3 generation turns TabZIP14 gene Arabidopsis leaf and all keeps
Green；After 200mM NaCl processing, in T3 generation, turns the root long of TabZIP14 gene arabidopsis L1, L2 and L3 strain than being wild respectively
The blade almost all of 1.2 times, 1.3 times and 1.3 times of type plant root long, WT lines is in albinism, and albefaction rate is
98% ± 0.4%, and in T3 generation, turns TabZIP14 gene Arabidopsis leaf and green state is presented substantially.These results suggest that turning
The salt-resistance of TabZIP14 gene arabidopsis significantly improves.TabZIP14 albumen and its encoding gene provided by the present invention are mentioning
It is of great significance, will be sent out in cultivating high resistance to cold and diseases such as strong frost resistance and strong salt-resistance plant variety in terms of high stress resistance of plant
Wave important function.
Detailed description of the invention
Fig. 1 is the relative expression quantity that PEG coerces lower wheat TabZIP14 gene.
Fig. 2 is the relative expression quantity of wheat TabZIP14 gene under salt stress.
Fig. 3 is the relative expression quantity of wheat TabZIP14 gene under low temperature stress.
Fig. 4 is the relative expression quantity that ABA coerces lower wheat TabZIP14 gene.
Fig. 5 is the rna level qualification result for turning TabZIP14 gene arabidopsis in T3 generation.
Fig. 6 is the phenotype of arabidopsis under low temperature stress.
Fig. 7 is the phenotype of arabidopsis under salt stress.
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.
KOD-Plus high fidelity enzyme: TOYOBO Products, catalog number KOD-201.
Entry vector pDONR/Zeo:invitrogen Products, catalog number 12535-035.
II Enzyme Mix:invitrogen Products of BP ClonaseTM, catalog number 11789-013.
Destination carrier pEarleyGate 100: in document " Zhang L, Zhang L, Xia C, Zhao G, Liu J, Jia
J,Kong X.A novel wheat bZIP transcription factor,TabZIP60,confers multiple
abiotic stress tolerances in transgenic Arabidopsis.Physiol Plant.2015,153
(4): being disclosed in 538-554. ", the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science.
II Enzyme Mix:invitrogen Products of LR ClonaseTM, catalog number 11791-019.
Agrobacterium GV3101-pMP90: in document " Xu Z, Kim S, Hyeon D, Kim D, et al.The
Arabidopsis thaliana NAC transcription factor family:structure–function
relationships and determinants of ANAC019 stress signaling.Biochem.J.2010,
426:183-196. " in be disclosed, the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science.
Arabidopsis (Arabidopsis thaliana) (Columbia-0 hypotype): document " He Y, Li W, Lv J,
Jia Y,Wang M and Xia G.Ectopic expression of a wheat MYB transcription factor
gene,TaMYB73,improves salinity stress tolerance in Arabidopsis thaliana.J Exp
Bot, 2012,63 (3): being disclosed in 1511-1522. ", and the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science.
Chinese spring (Chinese Spring Wheat): document " Kobayashia F, Maetaa E,
Terashimaa A,Takum S.Positive role of a wheat HvABI5ortholog in abiotic
It is disclosed in stress response of seedlings.Physiol Plant, 2008,134:74-86. ", the public can be from
Institute of Crop Science, Chinese Academy of Agricultural Science obtains.
The discovery of embodiment 1, TabZIP14 albumen and its encoding gene
In order to study function of the wheat bZIP gene in terms of plant stress-resistance, by the method for bioinformatics, from invention
22 encoding wheat bZIP are filtered out in the Wheat Full-length cDNA sequence for 30000 nonredundancies that laboratory where people has been sequenced
The gene of albumen.By the method for semiquantitive PCR, expression pattern of the 22 wheat bZIP genes under adverse environmental factor is carried out
Research, filters out the TabZIP14 gene by adverse circumstance inducing expression.
TabZIP14 full length gene cDNA is 1576bp, and sequence is as shown in sequence 2 in sequence table.Its open reading frame is sequence
The 181-1383 nucleotide from 5 ' ends of column 2, TabZIP14 albumen shown in sequence 1 in polynucleotide, sequence 1 are total
It is made of 400 amino acid residues.
The transcriptional level expression pattern of embodiment 2, TabZIP14 gene under different environment stresses
One, expression pattern of the TabZIP14 gene under PEG stress
The Chinese spring seedling of one heart stage of two leaves is placed in the PEG6000 aqueous solution of 16.1g/100ml and is handled,
Respectively before treatment (0h), processing after 1,3,6,12,24 and 48 hour extract wheat seedling root tissue RNA, reverse transcription at
CDNA is primer with upstream primer 1 and downstream primer 2 using its cDNA as template, carries out Real-time PCR Analysis, obtains
The relative expression quantity of TabZIP14 gene, internal reference are wheat Tubulin genes.
Primer for detecting TabZIP14 gene is as follows:
Upstream primer 1:5 '-CGGCTTGCTGATGTTAATC-3 '；
Downstream primer 2:5 '-TCGCTTGTGGAGAAGTAAG-3 '.
Primer for detecting internal reference Tubulin gene is as follows:
Tubulin-F:5 '-TTAGACTTGCGAAGCCAGCA-3 '；
Tubulin-R:5 '-AAATGCCCTTGAGGTTTCCC-3 '.
As a result as shown in Figure 1, TabZIP14 gene response PEG stress-inducing as seen from the figure.
Two, expression pattern of the TabZIP14 gene under salt stress
The Chinese spring seedling of one heart stage of two leaves is placed in 250mM NaCl aqueous solution and is handled, is being handled respectively
Before (0h), processing after 1,3,6,12,24 and 48 hour extract wheat seedling root tissue RNA, reverse transcription is at cDNA, with its cDNA
It is primer with upstream primer 1 and downstream primer 2 for template, carries out Real-time PCR Analysis, obtain the phase of TabZIP14 gene
To expression quantity, internal reference is wheat Tubulin gene.Specific primer sequence is referring to step 1.
As a result as shown in Fig. 2, TabZIP14 gene response salt stress induces as seen from the figure.
Three, expression pattern of the TabZIP14 gene under low temperature stress
4 DEG C of processing are placed in the Chinese spring seedling of one heart stage of two leaves, respectively before treatment 1 after (0h), processing, 3,6,
12, the RNA of wheat seedling Leaf tissue is extracted within 24 and 48 hours, reverse transcription is at cDNA, and using its cDNA as template, it is fixed in real time to carry out
PCR analysis is measured, obtains the relative expression quantity of TabZIP14 gene, internal reference is wheat Tubulin gene.Specific primer sequence referring to
Step 1.
As a result as shown in figure 3, TabZIP14 gene response low temperature stress induces as seen from the figure.
Four, expression pattern of the TabZIP14 gene under ABA stress
The Chinese spring seedling of one heart stage of two leaves is placed in 200 μM of ABA aqueous solutions and is handled, is being handled respectively
Before (0h), processing after 1,3,6,12,24 and 48 hour extract wheat seedling root tissue RNA, reverse transcription is at cDNA, with its cDNA
It is primer with upstream primer 1 and downstream primer 2 for template, carries out Real-time PCR Analysis, obtain the phase of TabZIP14 gene
To expression quantity, internal reference is wheat Tubulin gene.Specific primer sequence is referring to step 1.
As a result as shown in figure 4, TabZIP14 gene response ABA inducing expression as seen from the figure.
Embodiment 3, the acquisition for turning TabZIP14 gene plant and resistance of reverse identification
One, over-express vector is constructed using Gateway technology
1, attB design of primers
Upstream primer 3:5 '-GCATGGAGCGCGGCGTCTTC-3 '
Downstream primer 4:5 '-CTACGAGGTCGATCCGGATG-3 '
2, according to the needs of Gateway technology construction of expression vector, distinguish at the end 5' of upstream primer 3 and downstream primer 4
AttB1 and attB2 recombination site (underscore mark part is attB1 and attB2 recombination site) is added, respectively obtains upstream and draws
Object 5 and downstream primer 6.
Upstream primer 5:5 '-GGGGACAAGTTTGTACAAAAAAGCAGGCTGCATGGAGCGCGGCGTCTTC-3’
Downstream primer 6:5 '-GGGGACCACTTTGTACAAGAAAGCTGGGTCTACGAGGTCGATCCGGATG-3’
3, using the cDNA of Chinese spring as template, PCR amplification is carried out with upstream primer 5 and downstream primer 6, obtains attB
PCR product carries out PCR reaction using KOD-Plus high fidelity enzyme for the fidelity for guaranteeing PCR process, PCR reaction system and anti-
Answer program as follows:
4, BP recombining reaction is carried out according to following system and program, obtains BP reaction product:
5, the acquisition of introduction plasmid
50 μ l TOP10 competent cells are added in 2.5 μ l BP reaction products to convert, ice bath 30min, 42 DEG C of thermal shocks
Then 90s is immediately placed in 2min on ice.500 μ l LB culture solutions are added, 37 DEG C, 210rpm/min recovery 50min, resuscitation fluid is equal
The even solid LB Selective agar medium surface being coated on added with Zeocin (concentration is 40 μ g/ml), 37 DEG C of inversion overnight incubations.
pDONRTM/ Zeo carrier itself has lethal gene, cannot survive on culture medium.Pass through the BP recombining reaction target base of step 4
Because segment can replace lethal gene, finally obtained clone is entry clones (entry clone), and plasmid is introduction plasmid,
Send introduction plasmid to sequencing, as a result correctly.
6, LR recombining reaction is carried out according to following system and program, obtains LR reaction product:
7, the acquisition of target plasmid
Zeocin is only replaced with kanamycins (concentration is 50 μ g/ml) with step 5 by conversion process.
100 carrier of pEarleyGate, will be by target by LR recombining reaction lethal gene again with lethal gene
Genetic fragment replaces, and obtained clone is target clone (destination clone), and plasmid is target plasmid, by target
Plasmid is named as pEarleyGate 100-TabZIP14.The sequencing result of pEarleyGate 100-TabZIP14 shows
DNA fragmentation shown in 181-1383 containing sequence 2 in ordered list of pEarleyGate 100-TabZIP14.
PEarleyGate 100-TabZIP14 is TabZIP14 expression vector, and promoter is cauliflower mosaic virus 35S starting
Son.
Two, turn the acquisition of TabZIP14 gene arabidopsis
1, the target plasmid for obtaining step 1 converts Agrobacterium GV3101-pMP90, obtains recombinational agrobacterium.
2, arabidopsis infects and transgenic plant screening and identification
(1) culture of arabidopsis
Arabidopsis (Arabidopsis thaliana) (Columbia-0 hypotype) seed aqua sterilisa (is contained into volume hundred
Point content is the aqueous solution of 10% sodium hypochlorite and 10% Tween-20) disinfection 15min is shaken, in superclean bench with going out
Bacterium water cleans seed at least 5 times of above-mentioned disinfection.The seed cleaned is uniformly sowed on MS culture medium.MS plate is in 4 DEG C
Vernalization 3d is subsequently placed in 22 DEG C of illumination box cultures one week.It transplants after seedling grows four true leaves and is cultivated into nutritive cube,
Moisturizing 2-3d.The growth of arabidopsis is more sensitive to temperature, and 20-22 DEG C is than convenient cultivation temperature.Work as Arabidopsis plant
Grow to most of bud be in i.e. by bloom when, progress Agrobacterium infect.
(2) arabidopsis infects
It is infected, will be infected using the arabidopsis that the resulting recombinational agrobacterium of Hua Fayong step 1 obtains step (1) is stained with
The arabidopsis crossed is lain against in pallet, and dark moisturizing culture for 24 hours, is then put under regular culture conditions and cultivates.Arabidopsis infects 1
It can be infected again to improve transformation efficiency after week according to the growth conditions of arabidopsis, obtain turning TabZIP14 gene arabidopsis.
(3) the positive preliminary screening for turning TabZIP14 gene arabidopsis
1. collection turns TabZIP14 gene arabidopsis T0 for seed, (6-8d) is dried in 37 DEG C of baking ovens, then 4 DEG C of vernalization
3d。
2. seed is directly sown in nutritive cube, 22 DEG C moisturizing culture 1 week.
3. carrying out the positive after seedling grows 4 true leaves by sprinkling herbicide (basta) and turning the quasi- south of TabZIP14 gene
Mustard screening, the non-positive turns TabZIP14 gene arabidopsis and starts to occur wilting and stop growing after sprinkling 3 days, basic after 2 weeks
It is dead.It is thoroughly cleared in order to which the non-positive is turned TabZIP14 gene arabidopsis seedling, it can continuously spray 2-3 times, every minor tick 2-3d.
(4) the positive identification for turning TabZIP14 gene arabidopsis
The identification of A.DNA level
The positive that CTAB method extraction step (3) preliminary screening obtains turns the genome of TabZIP14 gene Arabidopsis leaf
DNA carries out PCR amplification with the forward primer 7 of TabZIP14 gene specific and reverse primer 8, obtains PCR using it as template
Pcr amplification product is carried out agarose gel electrophoresis by amplified production.
Forward primer 7:5 '-CAGAAGATGATGATATGGAAGG-3 '；
Reverse primer 8:5 '-TCGCTTGTGGAGAAGTAAG-3 '.
Electrophoresis result show step (3) preliminary screening obtain the positive turn TabZIP14 gene arabidopsis PCR amplification it
There is the purpose band of about 445bp afterwards, is further determined as turning TabZIP14 gene arabidopsis.Wildtype Arabidopsis thaliana is carried out above-mentioned
Identification experiment is without purpose band.
It is identified in aforementioned manners, turns TabZIP14 gene arabidopsis (hereinafter referred to as T3 generation for homozygosis until obtaining T3
Turn TabZIP14 gene arabidopsis), from gained T3 generation turn to randomly select 3 each strains in TabZIP14 gene arabidopsis, be denoted as L1,
L2 and L3.
The identification of B.RNA level
The T3 obtained to basta herbicide screening randomly selects three plants, extracts total serum IgE, go forward side by side for homozygous transgenic plant
The synthesis of the first chain of row cDNA, is primer with upstream primer 1 and downstream primer 2, carries out Real-time PCR Analysis, obtains
The relative expression quantity of TabZIP14 gene, internal reference are wheat Tubulin genes.Specific primer sequence is referring to 2 step 1 of embodiment.
As the result is shown: turning the T3 of TabZIP14 gene for homozygous plants, detected using RT-qPCR, the results showed that it is homozygous
The expression of TabZIP14 gene is close in strain L1, L2 and L3.It can continue follow-up function identification.Turn TabZIP14 gene
L1, L2 and L3 strain rna level qualification result it is specifically as shown in Figure 5.
Three, turn the degeneration-resistant identification of TabZIP14 gene arabidopsis
1, frost resistance is analyzed
The seed that wildtype Arabidopsis thaliana (WT) and three T3 generations turn TabZIP14 gene arabidopsis (L1, L2, L3) is existed
It 22 DEG C, is cultivated under 12h illumination, obtains 3 weeks seedling, then handled each seedling 3 hours at -10 DEG C, then renewal cultivation 4
It.Observation the cold treatment of each group arabidopsis before and after growth of seedling state and count its survival rate.Experiment in triplicate, repeats every time
12 plants of each strain.
The growth conditions of each seedling before and after cold treatment are as shown in fig. 6, as seen from the figure, with wildtype Arabidopsis thaliana phase
Than the frost resistance for turning TabZIP14 gene arabidopsis significantly improves.After freeze proof processing, the survival rate of WT lines is 16%,
The strain survival rate for turning TabZIP14 is 79% (the survival rate mean values of these three strains of L1, L2 and L3).
2, salt-resistance is analyzed
The seed that wildtype Arabidopsis thaliana (WT) and three T3 generations turn TabZIP14 gene arabidopsis (L1, L2, L3) is existed
It 22 DEG C, is vertically cultivated under 12h illumination, obtains 5 days seedling, the MS that each seedling is transferred to the NaCl containing 150 and 200mM is trained
It supports and is vertically cultivated on base, and be respectively control with the seedling vertically cultivated on the MS culture medium without NaCl.Observation is each
Organizing Arabidopsis thaliana Seedlings growth conditions and counting plant root long and plant albefaction rate (has the plant strain of albefaction blade after salt stress processing
It is several to handle the ratio between preceding plant strain number with salt stress).Experiment in triplicate, repeats 8 plants of each strain every time.
The state of each group Arabidopsis thaliana Seedlings is as shown in fig. 7, as seen from the figure, after 150mM NaCl processing, wild type is quasi-
The root long of southern mustard is obviously suppressed, and it is wild respectively that T3 generation, which turns the root long of TabZIP14 gene arabidopsis L1, L2 and L3 strain,
1.4 times, 1.3 times and 1.4 times of type plant root long.Albinism is presented in the arabidopsis leaf of Part Wild type, and albefaction rate is
37% ± 0.5%.And in T3 generation, turns TabZIP14 gene Arabidopsis leaf and all keeps green.After 200mM NaCl processing, T3 generation
Turn the root long of TabZIP14 gene arabidopsis L1, L2 and L3 strain than be respectively 1.2 times, 1.3 times of WT lines root long and
1.3 again.The blade almost all presentation albefaction of WT lines is present, and albefaction rate is 98% ± 0.4%；And in T3 generation, turns
Green state is presented in TabZIP14 gene Arabidopsis leaf substantially.These results suggest that turning the salt resistance of TabZIP14 gene arabidopsis
Property significantly improves.
Claims (13)
1. protein, the protein that the amino acid sequence shown in sequence 1 in sequence table forms.
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 are following DNA molecular 1) or 2):
1) DNA molecular shown in sequence 2 in sequence table；
2) DNA molecular shown in 181-1383 of sequence 2 in sequence table.
4. the recombinant vector containing nucleic acid molecules described in claim 2.
5. the recombinant vector containing nucleic acid molecules described in claim 3.
6. the expression cassette containing nucleic acid molecules described in Claims 2 or 3.
7. the recombinant microorganism containing nucleic acid molecules described in Claims 2 or 3.
8. recombinant vector according to claim 5, it is characterised in that: the recombinant vector is recombinant expression carrier or recombination
Cloning vector；
The promoter for starting the genetic transcription in the recombinant expression carrier is 35S promoter.
9. weight described in nucleic acid molecules described in protein or Claims 2 or 3 described in claim 1 or claim 4 or 5 or 8
Group carrier or expression cassette as claimed in claim 6 or recombinant microorganism as claimed in claim 7 it is following it is any in application:
(a) regulate and control stress resistance of plant；
(b) plant variety that breeding resistance improves；
The resistance is at least one of following: frost resistance, salt-resistance.
10. application according to claim 9, it is characterised in that: the plant is dicotyledon or monocotyledon.
11. the method for cultivating the genetically modified plants that resistance improves includes the steps that following (a1) and (a2):
(a1) encoding gene that protein described in claim 1 is imported into recipient plant, obtains expressing the encoding gene
Genetically modified plants；
(a2) it is obtained compared with the recipient plant from genetically modified plants obtained by step (a1), the transgenosis that resistance improves is planted
Object；
The resistance is at least one of following: frost resistance, salt-resistance.
12. according to the method for claim 11, it is characterised in that: in step (a1), the encoding gene is to pass through right
It is required that recombinant vector described in 4 or 5 or 8 imports the recipient plant.
13. according to the method for claim 11, it is characterised in that: the plant is dicotyledon or monocotyledon.
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| CN102747098A (en) * | 2011-04-21 | 2012-10-24 | 华中农业大学 | Application of modified gene OsbZIP46CA1 in control of draught resistance of rice |
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| CN102747098A (en) * | 2011-04-21 | 2012-10-24 | 华中农业大学 | Application of modified gene OsbZIP46CA1 in control of draught resistance of rice |
Non-Patent Citations (2)
| Title |
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| "AK360603.1";Matsumoto,T.等;《Genbank》;20110318;全文 * |
| "CAA56374.1";Vicente-Carbajosa,J.;《Genbank》;20061114;全文 * |
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