CN105037521B - A kind of and plant adversity resistance related protein TaWrky48 and its encoding gene and application - Google Patents
A kind of and plant adversity resistance related protein TaWrky48 and its encoding gene and application Download PDFInfo
- Publication number
- CN105037521B CN105037521B CN201510490829.9A CN201510490829A CN105037521B CN 105037521 B CN105037521 B CN 105037521B CN 201510490829 A CN201510490829 A CN 201510490829A CN 105037521 B CN105037521 B CN 105037521B
- Authority
- CN
- China
- Prior art keywords
- tawrky48
- sequence
- protein
- plant
- resistance
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Botany (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Gastroenterology & Hepatology (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Medicinal Chemistry (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a kind of and plant adversity resistance related protein TaWrky48 and its encoding gene and applications.Albumen relevant to stress resistance of plant provided by the present invention, is following protein a) or b) or c): a) amino acid sequence is protein shown in sequence 2 in sequence table；B) fused protein that the N-terminal of protein shown in sequence 2 and/or C-terminal connection label obtain in sequence table；C) protein with the same function for obtaining amino acid sequence shown in sequence 2 in sequence table by the substitution and/or deletion and/or addition of one or several amino acid residues.It is proved by test: the encoding gene of the invention with plant adversity resistance related protein TaWrky48 being imported into the drought resistance for turning TaWrky48 rice obtained after wild rice and is significantly higher than wild type, to degeneration-resistant new rice variety is cultivated, especially cultivation drought resisting rice has important theory and practical significance.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a kind of and plant adversity resistance related protein TaWrky48 and its volume
Code gene and application.
Background technique
In natural environment it is arid, saline and alkaline to plant growth and development have great influence, it is big to will cause crops when serious
The scale underproduction, cultivating resistance crop is one of main target of planting industry.Currently, it is obtained by genetic engineering breeding with resistance to
The crop varieties of inverse property are a kind of effective methods.And the technical bottleneck problem of most critical is effective degeneration-resistant base in this method
The screening of cause and function are found.
Transcription factor plays an important role during plant Stress response, they being capable of the degeneration-resistant phase of activated plant
The expression of correlation gene and the synthesis of different function albumen, cause various biochemical reactions.In addition, there is article to be reported in wheat
Several transcription factors including TaBTF3, TabZIP60, TaMBF1, TaWRKYs, TaMYBs and TaNACs take part in wheat
The relevant responsing reaction of abiotic stress.And the overexpression of some transcription factors effectively raises wheat in Different stress item
The resistance of part.
Summary of the invention
The technical problem to be solved by the present invention is to how regulate and control the resistance of plant.
In order to solve the above technical problems, being named as TaWrky48 albumen present invention firstly provides a kind of protein.
TaWrky48 albumen provided by the invention is following protein a) or b) or c):
A) amino acid sequence is protein shown in sequence 2 in sequence table；
B) fused protein that the N-terminal of protein shown in sequence 2 and/or C-terminal connection label obtain in sequence table；
C) by amino acid sequence shown in sequence 2 in sequence table by one or several amino acid residues substitution and/or
Obtained protein relevant to stress resistance of plant is deleted and/or added.
Wherein, amino acid sequence shown in sequence 2 is made of 137 amino acid residues in sequence table.
In order to make protein in a) convenient for purifying, can in sequence table the amino terminal of protein shown in sequence 2 or
Carboxyl terminal connects upper label as shown in Table 1.
The sequence of table 1, label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| Poly-His | 2-10 (usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tagⅡ | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
It is above-mentioned c) in protein in, the substitution and/or deletion and/or addition of one or several amino acid residues are
No more than the substitution and/or deletion and/or addition of 10 amino acid residues.
It is above-mentioned c) in protein can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression and obtain.
It is above-mentioned c) in the encoding gene of protein can be by the way that one will be lacked in DNA sequence dna shown in sequence 1 in sequence table
The codon of a or several amino acid residues, and/or the missense mutation of one or several base-pairs is carried out, and/or at its 5 ' end
And/or 3 ' end connect the coded sequence of label shown in table 1 and obtain.
In order to solve the above technical problems, the present invention also provides biomaterials relevant to above-mentioned TaWrky48 albumen.
Biomaterial relevant to above-mentioned TaWrky48 albumen provided by the invention is following A 1) any into A20)
Kind:
A1 the nucleic acid molecules of above-mentioned TaWrky48 albumen) are encoded；
A2) contain A1) expression cassettes of the nucleic acid molecules；
A3) contain A1) recombinant vectors of the nucleic acid molecules；
A4) contain A2) recombinant vector of the expression cassette；
A5) contain A1) recombinant microorganisms of the nucleic acid molecules；
A6) contain A2) recombinant microorganism of the expression cassette；
A7) contain A3) recombinant microorganism of the recombinant vector；
A8) contain A4) recombinant microorganism of the recombinant vector；
A9) contain A1) the transgenic plant cells systems of the nucleic acid molecules；
A10) contain A2) the transgenic plant cells system of the expression cassette；
A11) contain A3) the transgenic plant cells system of the recombinant vector；
A12) contain A4) the transgenic plant cells system of the recombinant vector；
A13) contain A1) Transgenic plant tissues of the nucleic acid molecules；
A14) contain A2) Transgenic plant tissue of the expression cassette；
A15) contain A3) Transgenic plant tissue of the recombinant vector；
A16) contain A4) Transgenic plant tissue of the recombinant vector；
A17) contain A1) the genetically modified plants organs of the nucleic acid molecules；
A18) contain A2) the genetically modified plants organ of the expression cassette；
A19) contain A3) the genetically modified plants organ of the recombinant vector；
A20) contain A4) the genetically modified plants organ of the recombinant vector.
In above-mentioned biomaterial, A1) nucleic acid molecules be it is following 1) or 2) or 3) shown in gene:
1) its coded sequence is the cDNA molecule or DNA molecular of sequence 1 in sequence table；
2) there is 75% or 75% or more identity with the nucleotide sequence 1) limited, and encodes the cDNA of above-mentioned protein
Molecule or genomic DNA molecule；
1) or 2) 3) and the cDNA molecule of above-mentioned protein is encoded with the nucleotide sequence hybridization that limits under strict conditions
Or genomic DNA molecule.
Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also
To be RNA, such as mRNA or hnRNA.
Wherein, nucleotide sequence shown in sequence 1 is made of 414 nucleotide in sequence table, sequence 2 in polynucleotide
Shown in amino acid sequence.
Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation
Method is mutated the nucleotide sequence of coding TaWrky48 albumen of the invention.Those by manually modified, have and this
The nucleotide sequence 75% of isolated TaWrky48 albumen or the nucleotide of higher identity are invented, as long as coding
TaWrky48 albumen and there is above-mentioned protein function, is derived from nucleotide sequence of the invention and being equal to the present invention
Sequence.
Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair
The nucleotide sequence for the protein that amino acid sequence shown in sequence 2 forms in bright polynucleotide has 75% or higher,
Or 85% or higher or 90% or higher or 95% or higher identity nucleotide sequence.Identity can with the naked eye or meter
Calculation machine software is evaluated.Using computer software, the identity between two or more sequences can use percentage (%) table
Show, can be used to evaluate the identity between correlated series.
Above-mentioned 75% or 75% or more identity can be 80%, 85%, 90% or 95% or more identity.
In above-mentioned biomaterial, A2) described in the nucleic acid molecules containing coding TaWrky48 albumen expression cassette, refer to energy
Enough DNA that TaWrky48 albumen is expressed in host cell, the DNA not only may include the starting for starting TaWrky48 genetic transcription
Son may also include the terminator for terminating TaWrky48 genetic transcription.Further, the expression cassette may also include enhancer sequence.
Promoter for use in the present invention includes but is not limited to: constitutive promoter, organizes, the promoter that organ and development are special, and
Inducible promoter.The example of promoter includes but is not limited to: the constitutive promoter 35S of cauliflower mosaic virus: coming westerly
The wound-inducible promoter of red persimmon, leucine aminopeptidase (" LAP ", Chao et al. (1999) Plant Physiol 120:
979-992)；Chemical inducible promoter from tobacco, pathogenesis correlation 1 (PR1) is (by salicylic acid and BTH (benzo thiophene two
Azoles -7- carbothioic acid S-methyl ester) induction)；Tomato protease inhibitors II promoter (PIN2) or LAP promoter are (available
Methyl jasmonate induction)；Heat-shock promoters (United States Patent (USP) 5,187,267)；Tetracycline inducible promoter (United States Patent (USP)
5,057,422)；Seed specific promoters, such as Millet Seed specificity promoter pF128 (CN101063139B (Chinese patent
200710099169.7)), the special promoter of seed storage protein matter (for example, phaseolin, napin, oleosin and big
The promoter (Beachy et al. (1985) EMBO is J.4:3047-3053) of beans beta conglycin).They can be used alone
Or it is used in combination with other plant promoters.All references cited herein is cited in full text.Suitable tanscription termination
Son includes but is not limited to: Agrobacterium nopaline syntase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S are terminated
Son, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator.
The recombinant vector of the TaWrky48 expression casette can be contained with existing expression vector establishment.The plant table
It include double base agrobacterium vector and the carrier etc. that can be used for plant micropellet bombardment up to carrier.As pAHC25, pBin438,
PCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or
PCAMBIA1391-Xb (CAMBIA company) etc..The plant expression vector also may include 3 ' end non-translational regions of foreign gene
Domain, i.e., comprising polyadenylation signals and any other DNA fragmentation for participating in mRNA processing or gene expression.The polyadenylic acid letter
Number bootable polyadenylic acid is added to 3 ' ends of mRNA precursor, as Agrobacterium crown gall nodule induces (Ti) plasmid gene (such as nopaline
Synthase gene Nos), plant gene (such as soybean storage protein genes) 3 ' end transcription non-translational region all have similar functions.
When using gene constructed plant expression vector of the invention, enhancer, including translational enhancer or transcriptional enhancer also can be used,
These enhancer regions can be ATG initiation codon or neighboring region initiation codon etc., but must read with coded sequence
Frame is identical, to guarantee the correct translation of entire sequence.The source of the translation control signal and initiation codon be it is extensive,
Can be it is natural, be also possible to synthesis.Translation initiation region can come from transcription initiation region or structural gene.In order to just
In transgenic plant cells or plant are identified and screened, plant expression vector used can be processed, it can as being added
The coding expressed in plant can produce the enzyme of color change or gene (gus gene, luciferase genes of luminophor
Deng), the marker gene of antibiotic (if assigned the nptII gene to kanamycins and associated antibiotic resistance, assigns to herbicide
The bar gene of phosphinothricin resistance assigns the hph gene to antibiotic hygromycin resistance, and assigns to methotrexate resistance
Dhfr gene is assigned to the EPSPS gene of glyphosate) or (such as anti-herbicide base such as anti-chemical reagent marker gene
Cause), provide metabolism mannose ability mannose-6-phosphate isomerase gene.It, can not from the security consideration of genetically modified plants
Add any selected marker, transformed plant is directly screened with adverse circumstance.
In above-mentioned biomaterial, the carrier can be plasmid, sticking grain, bacteriophage or viral vectors.
In above-mentioned biomaterial, the microorganism can be yeast, bacterium, algae or fungi, such as Agrobacterium.
In above-mentioned biomaterial, the transgenic plant cells system, Transgenic plant tissue and genetically modified plants organ are equal
It does not include propagation material.
In an embodiment of the invention, the encoding gene of TaWrky48 albumen is (i.e. in sequence table shown in sequence 1
Nucleotide) it is imported by the recombinant vector pCUbi1390-TaWrky48 of the expression cassette of the encoding gene containing TaWrky48 albumen
In Agrobacterium EHA105.The recombinant vector pCUbi1390-TaWrky48 is to lead to DNA molecular shown in sequence 1 in sequence table
Cross the carrier obtained in LR reaction recombination to pCUbi1390 carrier, recombinant vector pCUbi1390-TaWrky48 expression
TaWrky48 albumen.
In order to solve the above technical problems, the present invention also provides above-mentioned TaWrky48 albumen or with above-mentioned TaWrky48 albumen
The purposes of relevant biomaterial.
The present invention provides above-mentioned TaWrky48 albumen or biomaterial relevant to above-mentioned TaWrky48 albumen to regulate and control
Application in stress resistance of plant；
The present invention also provides above-mentioned TaWrky48 albumen or biomaterial relevant to above-mentioned TaWrky48 albumen to train
Educate the application in resistance genetically modified plants.
In above-mentioned application, the regulation stress resistance of plant is to improve stress resistance of plant.
In above-mentioned application, the resistance is drought resistance and/or salt-resistance.
In above-mentioned application, the plant can be monocotyledon and/or dicotyledon；The monocotyledon specifically may be used
For rice (OryzasativaLcv.Nipponbare Nipponbare).
In order to solve the above-mentioned technical problem, the present invention finally provides a kind of genetically modified plants of cultivation resistance raising
Method.
A kind of method for cultivating the genetically modified plants that resistance improves provided by the invention includes by above-mentioned TaWrky48 egg
The step of white encoding gene imports in recipient plant, obtains genetically modified plants；The genetically modified plants resistance is higher than described
Recipient plant.
In the above method, the nucleotide sequence of the encoding gene of the TaWrky48 albumen is in sequence table shown in sequence 1
DNA molecular.
In the above method, the resistance is drought resistance and/or salt-resistance, and the drought resistance and/or salt-resistance specifically may be used
It is presented as under arid or salt stress, compared with recipient plant: (1) genetically modified plants are better than the upgrowth situation of recipient plant；
(2) genetically modified plants are higher than the survival rate of seedling of recipient plant；(3) genetically modified plants are lower than the seedling wilted percent of recipient plant；
(4) genetically modified plants are lower than the mda content of recipient plant.
In the above method, the recipient plant can be monocotyledon and/or dicotyledon；The monocotyledon is
Rice (OryzasativaLcv.Nipponbare Nipponbare).
In an embodiment of the present invention, the encoding gene (nucleosides shown in sequence 1 i.e. in sequence table of TaWrky48 albumen
Acid) agriculture bar imported by the recombinant vector pCUbi1390-TaWrky48 of the expression cassette of the encoding gene containing TaWrky48 albumen
In bacterium EHA105.The recombinant vector pCUbi1390-TaWrky48 is that DNA molecular shown in sequence 1 in sequence table is passed through LR
The carrier obtained in reaction recombination to pCUbi1390 carrier, recombinant vector pCUbi1390-TaWrky48 express TaWrky48 egg
It is white.
In the above method, the TaWrky48 gene can be modified first as follows, then import in recipient plant, to reach more
Good expression effect:
1) it modifies and optimizes according to actual needs, so that gene efficient expression；For example, can be according to recipient plant institute partially
The codon of love changes its codon while keeping the amino acid sequence of TaWrky48 gene of the present invention to meet and plant
Object preferences；In optimization process, it is desirable that keep certain G/C content in the coded sequence after optimization, planted with being best implemented with
The high level expression of quiding gene in object, wherein G/C content can be 35%, be more than 45%, more than 50% or more than about 60%；
2) gene order of neighbouring initial methionine is modified, so that translation effectively starting；For example, using in plant
The effective sequence known is modified；
3) it is connect with the promoter of various plants expression, in favor of its expression in plant；The promoter may include
Composing type, induction type, timing adjusting, growth adjustment, Chemical Regulation, tissue are preferably and tissue-specific promoter；Promoter
Selection will need with expression time and space and be changed, and also depend on target kind；Such as the specificity of tissue or organ
Promoter is expressed, receptor as needed is depending on what period of development；Although demonstrating many from dicotyledon
Promoter can act in monocotyledon, and vice versa, but it is desirable to select dicot promoters are used for
Expression in dicotyledon, monocotyledonous promoter is for the expression in monocotyledon；
4) it is connect with suitable transcription terminator, can also be improved the expression efficiency of gene of the present invention；Such as from
The tml of CaMV, from the E9 of rbcS；Any known available terminator to work in plant can be with the present invention
Gene is attached；
5) enhancer sequence is introduced, such as intron sequences (such as from Adhl and bronzel) and viral leader sequence
(such as from TMV, MCMV and AMV).
The TaWrky48 gene recombinant vectors can be by using Ti-plasmids, plant virus carrying agent, and direct DNA turns
Change, microinjection, the standard biologics technical method such as electroporation imports plant cell.
In the above method, the genetically modified plants are interpreted as not only including by the TaWrky48 genetic transformation purpose plant
Obtained first generation genetically modified plants also include its filial generation.For genetically modified plants, the gene can be bred in the species,
The gene transfer can also be entered to other kinds of same species with traditional breeding techniques, particularly including in commercial variety.It is described
Genetically modified plants include seed, callus, intact plant and cell.
The nucleic acid molecules overall length of the above-mentioned TaWrky48 albumen of amplification coding or the primer pair of its segment also belong to of the invention
Protection scope.
The present invention has found the wheat transcription factor TaWrky48 albumen with significant degeneration-resistant border ability from wheat.It will
The experiment of TaWrky48 channel genes wild rice (OryzasativaLcv.Nipponbare) can prove that under salt stress, T2In generation, turns TaWrky48 base
Because the upgrowth situation of plant is better than wild type, mda content lower than wild type；Under drought stress, T2In generation, turns TaWrky48 base
Because the survival rate of seedling of plant is higher than wild type, seedling wilted percent is lower than wild type.Illustrate TaWrky48 base provided by the invention
Because the albumen encoded with it can significantly improve the drought resistance of rice, to degeneration-resistant new rice variety is cultivated, especially cultivation drought resisting
Rice has important theory and practical significance.
Detailed description of the invention
Fig. 1 is the structure chart of plant over-express vector pCUbi1390.
Fig. 2 is T2In generation, turns the Molecular Identification of TaWrky48 rice strain.Wherein, Fig. 2 a is T2In generation, turns TaWrky48 rice strain
The PCR of system is detected, wherein P is pCUbi1390 plasmid, and NT is to turn empty carrier plant, and 1-6 is 6 T2In generation, turns TaWrky48 rice
Strain；Fig. 2 b is 4 T2In generation, turns the RT-PCR expression analysis of TaWrky48 rice strain, wherein Actin1 is control, and NT is to turn
Empty carrier plant, 2-5 are 4 T2In generation, turns TaWrky48 rice strain.
Fig. 3 is T2In generation, turns the upgrowth situation of TaWrky48 rice strain before and after the processing in salt stress.The left side is at salt stress
The upgrowth situation of (0 day of NaCl) before reason；The right is the upgrowth situation of (10 day of NaCl) after salt stress is handled 10 days.Its
In, TaWrky48-2, TaWrky48-3, TaWrky48-4 are T2In generation, turns TaWrky48 rice strain, and Ctrl is to turn empty carrier
Plant.
Fig. 4 is T after Osmotic treatment2In generation, turns the statistical result of the survival rate of seedling of TaWrky48 rice strain.Wherein,
TaWrky48-2, TaWrky48-3, TaWrky48-4 are T2In generation, turns TaWrky48 rice strain, and Ctrl is to turn empty carrier plant
Strain.
Fig. 5 is T after Osmotic treatment 20 days2In generation, turns the statistical result of the seedling wilted percent of TaWrky48 rice strain.Wherein,
TaWrky48-2, TaWrky48-3 and TaWrky48-4 are T2In generation, turns TaWrky48 rice strain；Ctrl is to turn empty carrier plant
Strain.
Fig. 6 is NaCl T before and after the processing2In generation, turns the measurement result of the mda content of TaWrky48 rice strain.Wherein,
TaWrky48-2, TaWrky48-3, TaWrky48-4 T2In generation, turns TaWrky48 rice strain；Ctrl is to turn empty carrier plant.
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.
Wheat breed China spring in following embodiments is in document " history of " China spring " wheat, Acta Agronomica Sinica." in it is public
It opened, the public can obtain from Biological Technology institute, Chinese Academy of Agricultural Sciences.
Rice varieties OryzasativaLcv.Nipponbare Nipponbare in following embodiments is in document " Zou, 2011 [Leaf rolling
It is public in controlled by the homeodomain leucine zipper class IV gene Roc5 in rice. "
It opened, the public can obtain from Biological Technology institute, Chinese Academy of Agricultural Sciences.
Yoshida nutrient solution in following embodiments is made of solute and solvent, and solvent is water, solute and its
Concentration in Yoshida nutrient solution are as follows: NH4NO3116.0g/L、NaH2PO4·2H2O 49.9g/L、K2SO487.0g/L、
CaCl2111.0g/L、MgSO4·7H2O 418.0g/L、MnCl2·4H2O 1801.8mg/L、(NH4)6MoO24·4H2O
91.8mg/L、H3BO31098.0mg/L、ZnSO4·7H2O 44.55mg/L、CuSO4·5H2O 41.6mg/L、FeCl3·6H2O
9738.0mg/L, citric acid (hydrate) 14875.0mg/L.
The clone of embodiment 1, wheat TaWrky48 gene
1, the acquisition of cDNA
The RNA of wheat (China spring) is extracted, reverse transcription obtains cDNA.
2, PCR amplification
The cDNA obtained using step 1 carries out PCR amplification as template, using TaWrky48F and TaWrky48R primer, obtains
Pcr amplification product.Primer sequence is as follows:
TaWrky48F:5'-CTGACCACCGCCGTTTGA-3'(sequence 3)；
TaWrky48R:5'-ACAACAGGGAGCGGAGAAG-3'(sequence 4).
3, the acquisition of TaWrky48 gene
Pcr amplification product that agarose gel electrophoresis detecting step 2 obtains and sequencing: the result shows that PCR amplification obtain it is big
The small amplified fragments for 414bp length are named as TaWrky48 gene, the nucleotide sequence of TaWrky48 gene such as sequence
In table shown in sequence 1, sequence 1 is made of 414 nucleic acid molecules in sequence table；The albumen of TaWrky48 gene coding is named as
TaWrky48 albumen, the amino acid sequence of the albumen are the sequence 2 in sequence table；Sequence 2 is residual by 137 amino acid in sequence table
Base composition.
Embodiment 2, the acquisition for turning TaWrky48 rice and its resistance analysis
One, turn the acquisition of TaWrky48 trans-genetic hybrid rice
1, the acquisition of pCUbi1390-TaWrky48
(1) acquisition of TaWrky48 gene
The RNA of wheat (China spring) is extracted, reverse transcription obtains cDNA.Using the cDNA of acquisition as template, using TaWrky48F
PCR amplification is carried out with TaWrky48R primer, obtains pcr amplification product, i.e. TaWrky48 gene.Primer sequence is as follows:
TaWrky48F:5'-CTGACCACCGCCGTTTGA-3'(sequence 3)；
TaWrky48R:5'-ACAACAGGGAGCGGAGAAG-3'(sequence 4).
(2) the TaWrky48 gene for obtaining step (1) and pDONRTM/Zeo carrier (BP
II (Invitrogen)) BP reaction is carried out, obtain entry vector.
(3) entry vector that step (2) obtains is transformed into E.coli DH5 α cell through heat shock method, obtained gram
Grand is entry clones, and the plasmid in the entry clones is introduction plasmid.The introduction plasmid is named as pDONRTM/Zeo-
TaWrky48, and send introduction plasmid to sequencing, sequencing result shows the introduction plasmid containing DNA shown in sequence 1 in ordered list
Molecule.
(4) the introduction plasmid pDONRTM/Zeo-TaWrky48 for obtaining step (3) and purpose carrier pCUbi1390 (LRII (Invitrogen)) LR reaction is carried out, LR reaction product is obtained, as includes
The binary expression vector of TaWrky48 gene.
50 μ l TOP10 competent cells are added in 2.5 μ l LR reaction product obtained above to convert, obtained gram
Grand is target clone, and the plasmid in target clone is target plasmid, which is named as pCUbi1390-
TaWrky48, and sequence verification is carried out to it.
Sequencing result shows: recombinant plasmid pCUbi1390-TaWrky48 is will be shown in sequence 1 in sequence table
By LR reaction recombination to the carrier obtained on pCUbi1390 carrier, (Fig. 1, position shown in arrow are TaWrky48 gene
TaWrky48 gene is inserted into the position on carrier).PCUbi1390-TaWrky48 is TaWrky48 expression vector,
PCUbi1390-TaWrky48 contains TaWrky48 expression casette, in TaWrky48 expression casette, starts TaWrky48 base
Because the promoter of transcription is cauliflower mosaic virus 35 S promoter.
2, the acquisition of recombinational agrobacterium
Recombinant plasmid pCUbi1390-TaWrky48 is converted into Agrobacterium EHA105 with electric shocking method and (steps its biology purchased from Shanghai
Science and Technology Ltd., article No. CH5002B), which is named as EHA105/ by picking recombinational agrobacterium
pCUbi1390-TaWrky48。
According to the above method, recombinant plasmid pCUbi1390-TaWrky48 is replaced with into plasmid pCUbi1390, other steps are equal
It is identical, recombinational agrobacterium will be obtained and be named as EHA105/pCUbi1390.
3, turn the acquisition and identification of TaWrky48 rice
(1) it when the OD600 value of the agrobacterium suspension obtained when step 2 reaches 0.120~0.140, is infected with the bacterium solution
The callus of rice (OryzasativaLcv.Nipponbare Nipponbare) infects 30 minutes, during which uses hand jog frequently.Callus is transferred to
On subculture medium (table 2), it is protected from light co-cultivation 3~4 days；After co-cultivation, callus is transferred to subculture medium 5 days
It is transferred on Selective agar medium (table 2) that cultivate 10~15 days subsequent generations primary behind left and right；Picking is grown from former callus surface
New resistant calli out is transferred on pre- differential medium (table 2), differentiation culture 7 days in advance under 28 DEG C of dark；Selection
The smooth callus in off-white color, surface is transferred on differential medium (table 2), 28 DEG C of differentiation cultures: first cultivates 3 under dark
It, then cultivates 15~20 days under lasting cold light photograph；The seedling regenerated from resistant calli is transferred to culture of rootage
On base (table 2), continuous light culture 15 days, the good seedling of upgrowth situation can be transferred directly to field planting growth, finally
To 47 plants of T0In generation, turns TaWrky48 rice strain, by T1The seed that generation turns TaWrky48 rice continues to repeat the above steps, until
To T2In generation, turns TaWrky48 rice strain.
According to the above method, recombinational agrobacterium EHA105/pCUbi1390-TaWrky48 is replaced with into recombinational agrobacterium
EHA105/pCUbi1390, other steps are all the same, obtain T2In generation, turns empty carrier rice strain.
The formula of table 2, culture medium
(2) turn the Molecular Identification of TaWrky48 rice
Randomly choose 100 T2In generation, turns TaWrky48 rice strain, to turn empty carrier rice and wild rice (OryzasativaLcv.Nipponbare
It Nipponbare is) control, each strain takes about 500mg plant leaf CTAB method to extract genomic DNA, usesSpectrophotometer (Beckman, Fullerton, CA) and the method for gel electrophoresis quantify DNA sample, use
Takara PCR mix(Takara Bio Inc.,Otsu,Japan；Http:// www.takara.com.cn) and
PCUbi1390 carrier insertion point flank primers (Test-PF [5'-GAATTCTAAGAGGAGTCCACCATG-3'] and Test-
PR [5'-GAAATTCGAGCTGGTCACCTG-3']) carry out PCR detection.
As a result as shown in Figure 2 a: wild rice plant and turning empty carrier rice plant and do not expand after above-mentioned primer amplification
Increasing shaping band, and T2In generation, turns the band that the amplifiable size out of TaWrky48 rice strain is 357bp.Choose T2In generation, turns
TaWrky48 rice strain TaWrky48-2, TaWrky48-3, TaWrky48-4 are used for the research of following resistance experiment.
Two, turn the resistance analysis of TaWrky48 rice
By T2In generation, turns TaWrky48 rice strain TaWrky48-2, TaWrky48-3, TaWrky48-4, turns empty carrier plant
42 DEG C are placed in the seed of wild rice (OryzasativaLcv.Nipponbare Nipponbare) and saves one week with breaking dormancy, and seed exists in room temperature
It is impregnated 3 days in water, is subsequently placed at 37 DEG C and sprouts one day；Consistent seedling will be grown to be put into 96 orifice plates (cutting bottom),
It is impregnated under 37 DEG C of dark conditions and cultivates one day in water to promote root growth, be transferred in seedling-cultivation plate later and continue culture [24
℃(13h)/20℃(11h)]；After 5 days, changes water into Yoshida nutrient solution and continue to cultivate；After 7 days, respectively in salt stress nutrition
Liquid (the Yoshida nutrient solution containing 150mM NaCl) and drought stress the nutrient solution (PEG for being 20% containing mass fraction
Yoshida nutrient solution) middle culture 10 days.Observe T under the growing state and detection drought stress conditions under salt stress2In generation, turns
The expression of TaWrky48 gene in TaWrky48 rice strain.Each strain chooses 40 plants, all experimental techniques repeat and
Biology repeats each 3 times.
(1) detection of the upgrowth situation and mda content under salt stress
A, the T under salt stress2The growing state that generation turns TaWrky48 rice strain is as shown in Figure 3: can from figure
Out, (0 day of NaCl) T before salt stress processing2Generation turn TaWrky48 rice strain TaWrky48-2, TaWrky48-3,
TaWrky48-4 and turn the growing state of empty carrier plant without significant difference, but (the NaCl 10 after salt stress is handled 10 days
Day), T2In generation, turns TaWrky48 rice strain TaWrky48-2, TaWrky48-3, TaWrky48-4 and turns the life of empty carrier plant
Long situation, which is significantly better than, turns empty carrier plant.
There was no significant difference with empty carrier plant is turned for the growing state of WT lines.
B, malonaldehyde (MDA) is one of final decomposition product of Lipid peroxidation metabolism, and content can reflect plant and be hurt
Harmful degree.To salt stress treated T2Generation turn TaWrky48 rice strain TaWrky48-2, TaWrky48-3,
TaWrky48-4, turn empty carrier plant and the mda content of wild rice (OryzasativaLcv.Nipponbare) is measured, the specific side of measurement
Referring to document, " Wang Jingying, Ao Hong, Zhang Jie wait plant physiology and biochemistry experimental technique and principle [M], Harbin: Northeast forestry to method
University press, the method in 2003. ".
As a result as shown in Figure 6: it can be seen from the figure that after salt stress processing, T2In generation, turns TaWrky48 rice strain
TaWrky48-2, TaWrky48-3, TaWrky48-4, the MDA content for turning empty carrier plant are risen, all plant by
To the injury of different degrees of alkaline stress, but after salt stress is handled 10 days, T2Generation turn TaWrky48 rice strain TaWrky48-2,
The mda content of TaWrky48-3, TaWrky48-4, which are significantly lower than, turns empty carrier plant, T2In generation, turns TaWrky48 rice strain
The amplitude that the MDA content of TaWrky48-2, TaWrky48-3, TaWrky48-4 rise, which is substantially less than, turns empty carrier plant, explanation
Its membrane oxidation degree is lower, and the injury being forced is lighter, and TaWrky48 gene can the positive salt-resistance for adjusting rice.
(2) detection of the expression analysis under drought stress, survival rate of seedling and seedling wilted percent
A, it extracts respectively through drought stress treated T2Generation turn TaWrky48 rice strain TaWrky48-2,
The total serum IgE of TaWrky48-3, TaWrky48-4, the root for turning empty carrier seedling and wild rice (OryzasativaLcv.Nipponbare) and stem are used
FastQuant RT Kit(with gDNase；Tiangen Biotech, Beijing, China) reverse transcription acquisition cDNA, with
CDNA is template, carries out PCR amplification using TaWrky48F and TaWrky48R primer, detects lower turn of drought stress conditions
The expression of TaWrky48 gene in TaWrky48 rice strain.Using OsACTIN1 as internal reference, internal control primer sequence:
ActinF:5'-TGTATGCCAGTGGTCGTACCA-3'；
ActinR:5'-CCAGCAAGGTCGAGACGAA-3'；
TaWrky48F:5'-CTGACCACCGCCGTTTGA-3'；
TaWrky48R:5'-ACAACAGGGAGCGGAGAAG-3'。
Testing result is as shown in Figure 2 b: turn empty carrier plant (NT) without amplified band, and T2In generation, turns TaWrky48 rice strain
System can amplify purpose band, show that foreign gene TaWrky48 gene is not only smoothly integrated into the genome of rice
On, and normal transcription can be expressed in transgenic paddy rice.The testing result of WT lines and the nothing for turning empty carrier plant
Significant difference.
B, the detection of seedlings under drought stress survival rate
To through drought stress treated T2Generation turn TaWrky48 rice strain TaWrky48-2, TaWrky48-3,
TaWrky48-4, turn empty carrier plant and the survival rate of seedling of wild rice (OryzasativaLcv.Nipponbare) is detected, the specific side of detection
Referring to document, " Wang Jingying, Ao Hong, Zhang Jie wait plant physiology and biochemistry experimental technique and principle [M], Harbin: Northeast forestry to method
University press, the method in 2003. ".
The testing result of survival rate of seedling is as shown in Figure 5: as can be seen from the figure: after drought stress processing, T2In generation, turns
The survival rate of seedling of TaWrky48 rice strain TaWrky48-2, TaWrky48-3, TaWrky48-4, which are significantly higher than, turns empty carrier
Plant.
There was no significant difference with empty carrier plant is turned for the growing state of WT lines.
C, the detection of seedlings under drought stress wilted percent
To through drought stress treated T2Generation turn TaWrky48 rice strain TaWrky48-2, TaWrky48-3,
TaWrky48-4, turn empty carrier plant and the seedling wilted percent of wild rice (OryzasativaLcv.Nipponbare) is detected, the specific side of detection
Referring to document, " Wang Jingying, Ao Hong, Zhang Jie wait plant physiology and biochemistry experimental technique and principle [M], Harbin: Northeast forestry to method
University press, the method in 2003. ".
The testing result of seedling wilted percent is as shown in Figure 6: as can be seen from the figure: after drought stress processing, T2In generation, turns
The seedling wilted percent of TaWrky48 rice strain TaWrky48-2, TaWrky48-3, TaWrky48-4, which are substantially less than, turns empty carrier
Plant.
There was no significant difference with empty carrier plant is turned for the growing state of WT lines.
The variation of above each index illustrates that the resistance of rice can be improved in TaWrky48 albumen, has been improved particularly rice
Salt-resistance and drought resistance.
Claims (6)
1. protein is following protein a) or b):
A) amino acid sequence is protein shown in sequence 2 in sequence table；
B) fused protein that the N-terminal of protein shown in sequence 2 and/or C-terminal connection label obtain in sequence table.
2. it is following A 1 biomaterial relevant to protein described in claim 1) any one of to A8):
A1 the nucleic acid molecules of protein described in claim 1) are encoded；
A2) contain A1) expression cassettes of the nucleic acid molecules；
A3) contain A1) recombinant vectors of the nucleic acid molecules；
A4) contain A2) recombinant vector of the expression cassette；
A5) contain A1) recombinant microorganisms of the nucleic acid molecules；
A6) contain A2) recombinant microorganism of the expression cassette；
A7) contain A3) recombinant microorganism of the recombinant vector；
A8) contain A4) recombinant microorganism of the recombinant vector.
3. relevant biological material according to claim 2, it is characterised in that: A1) described its coded sequence of nucleic acid molecules is
The cDNA molecule or DNA molecular of sequence 1 in sequence table.
4. protein described in claim 1 or relevant biological material described in claim 2 or 3 are in regulation stress resistance of plant
Application；
Or protein described in claim 1 or relevant biological material described in claim 2 or 3 are cultivating resistance transgenosis
Application in plant；The resistance is salt-resistance and/or drought resistance；
The plant is rice.
5. a kind of method for cultivating the genetically modified plants that resistance improves, including by the coding of protein described in claim 1
In channel genes recipient plant, the step of obtaining genetically modified plants；The resistance of the genetically modified plants is planted higher than the receptor
Object；The resistance is salt-resistance and/or drought resistance；
The plant is rice.
6. according to the method described in claim 5, it is characterized by: the nucleotide sequence of the encoding gene of the protein is sequence
DNA molecular shown in sequence 1 in list.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510490829.9A CN105037521B (en) | 2015-08-11 | 2015-08-11 | A kind of and plant adversity resistance related protein TaWrky48 and its encoding gene and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510490829.9A CN105037521B (en) | 2015-08-11 | 2015-08-11 | A kind of and plant adversity resistance related protein TaWrky48 and its encoding gene and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105037521A CN105037521A (en) | 2015-11-11 |
| CN105037521B true CN105037521B (en) | 2019-02-12 |
Family
ID=54444532
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510490829.9A Expired - Fee Related CN105037521B (en) | 2015-08-11 | 2015-08-11 | A kind of and plant adversity resistance related protein TaWrky48 and its encoding gene and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105037521B (en) |
Families Citing this family (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106854239B (en) * | 2015-12-09 | 2020-08-25 | 中国科学院遗传与发育生物学研究所 | Wheat stripe rust resistance related protein TaSPX-MFS and coding gene and application thereof |
| CN106866804B (en) * | 2015-12-11 | 2021-03-05 | 中国科学院植物研究所 | CAM01 protein related to plant photosynthesis and encoding gene and application thereof |
| CN107338230B (en) * | 2016-05-03 | 2019-09-13 | 中国科学院遗传与发育生物学研究所 | The application of OsMPK11 albumen and its encoding gene in regulation plant drought resistance |
| CN107338231B (en) * | 2016-05-03 | 2020-04-28 | 中国科学院遗传与发育生物学研究所 | Application of OsMPK21-1 protein and coding gene thereof in regulation and control of plant drought resistance |
| CN108424438B (en) * | 2018-05-17 | 2021-06-25 | 青岛大学 | Wheat powdery mildew resistance-related protein TaWRKY49, and coding gene and application thereof |
| CN110713526B (en) * | 2018-06-27 | 2021-05-18 | 中国农业科学院作物科学研究所 | Wheat stress-resistant protein TaBZR2D and coding gene and application thereof |
| CN111205355B (en) * | 2018-11-21 | 2021-08-13 | 中国农业科学院作物科学研究所 | Plant stress tolerance related protein SiWRKY76 and coding gene and application thereof |
| CN111285927B (en) * | 2018-11-21 | 2021-07-27 | 中国农业科学院作物科学研究所 | Plant stress tolerance related protein SiWRKY78 and coding gene and application thereof |
| CN112831505B (en) * | 2021-03-16 | 2023-04-11 | 昆明理工大学 | Pseudo-ginseng WRKY transcription factor gene PnWRKY15 and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399268A (en) * | 2010-09-10 | 2012-04-04 | 中国科学院遗传与发育生物学研究所 | Plant stress tolerance-related transcription factor GmNAC11, coding gene and application thereof |
| CN104059137A (en) * | 2013-03-21 | 2014-09-24 | 中国科学院遗传与发育生物学研究所 | GsNAC74 and application of its encoding gene in cultivation of stress tolerance plant |
-
2015
- 2015-08-11 CN CN201510490829.9A patent/CN105037521B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102399268A (en) * | 2010-09-10 | 2012-04-04 | 中国科学院遗传与发育生物学研究所 | Plant stress tolerance-related transcription factor GmNAC11, coding gene and application thereof |
| CN104059137A (en) * | 2013-03-21 | 2014-09-24 | 中国科学院遗传与发育生物学研究所 | GsNAC74 and application of its encoding gene in cultivation of stress tolerance plant |
Non-Patent Citations (2)
| Title |
|---|
| GenBank：AFV67804.1;Meng，Y.;《GenBank》;20121106;全文 * |
| HvWRKY10, HvWRKY19, and HvWRKY28 regulate Mla-triggered immunity and basal defense to barley powdery mildew;Meng Y等;《Mol Plant Microbe Interact》;20121130;第25卷(第11期);摘要 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105037521A (en) | 2015-11-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105037521B (en) | A kind of and plant adversity resistance related protein TaWrky48 and its encoding gene and application | |
| CN105753953B (en) | Disease-resistant wheat albumen and encoding gene and its application in regulation disease resistance of plant | |
| CN106188257B (en) | The application of soybean transcription factor GmbZIP336 and its encoding gene in regulation seed grain weight | |
| CN109111514A (en) | And the breeding method and its relevant biological material of the transgenic wheat of anti-banded sclerotial blight and root rot | |
| CN107459565A (en) | Application of the soybean drought resisting GAP-associated protein GAP in regulating and controlling soybean drought resistance | |
| CN110078807A (en) | Promote the protein and its encoding gene of potassium ion efficient absorption | |
| CN105218650B (en) | A kind of and plant adversity resistance related protein Prp1 and its encoding gene and application | |
| CN105061569B (en) | A kind of and relevant SiMYB107 albumen of stress resistance of plant and its relevant biological material and application | |
| CN107629121A (en) | One from transcription factor ZmNLP9 of corn and application thereof | |
| CN105111295B (en) | The breeding method and its relevant biological material for turning WMYB-R DNA triticums of anti-root rot and banded sclerotial blight | |
| CN109971766A (en) | A kind of and plant stress tolerance-associated protein PwRBP1 and its encoding gene and application | |
| CN107602683B (en) | Transcription factor ZmNLP4 from corn and application thereof | |
| CN105585623B (en) | The disease-resistant breeding method for turning TaMYB-KW DNA triticum and relevant biological material and application | |
| CN105713079B (en) | Protein and its relevant biological material are improving the application in plant products | |
| CN106047833B (en) | The application of OsCIPK31 and its encoding gene in regulation plant herbicide resistance | |
| CN105820220B (en) | The application of resistance relevant protein and its encoding gene in regulation plant alkali resistance | |
| CN105985417B (en) | Application of the growth associated protein GRP2 in regulating plant growth | |
| CN106367433B (en) | Plant is improved to the method and its application of gibberellin inhibitor sensitiveness | |
| CN105504031B (en) | From the grain weight GAP-associated protein GAP and its relevant biological material of soybean and application | |
| CN107663230A (en) | Application of the cold-resistant GAP-associated protein GAP in plant cold tolerance is regulated and controled | |
| CN105985419B (en) | Application of the growth associated protein GRP1 in regulating plant growth | |
| CN105985418B (en) | Application of the growth associated protein GRP4 in regulating plant growth | |
| CN105646683B (en) | The application of complete salt tolerant protein matter and relevant biological material in regulation plant salt endurance | |
| CN104744579B (en) | Applications of the resistance relevant protein GmL16 in regulating and controlling stress resistance of plant | |
| WO2022055751A1 (en) | Plastid transformation by complementation of nuclear mutations |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20190212 Termination date: 20210811 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |