CN105254727B - A kind of relevant transcription factor of hybridization paper mulberry drought stress and its encoding gene and application - Google Patents
A kind of relevant transcription factor of hybridization paper mulberry drought stress and its encoding gene and application Download PDFInfo
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Abstract
The invention discloses a kind of relevant transcription factor of hybridization paper mulberry drought stress and its encoding gene and applications.The hybridization relevant transcription factor of paper mulberry drought stress provided by the present invention is following protein a) or b) or c)：A) amino acid sequence is protein shown in sequence 1 in sequence table；B) fused protein that the N-terminal of protein shown in sequence 1 and/or C-terminal connection label obtain in sequence table；C) by amino acid sequence shown in sequence in sequence table 1 by the substitution of one or several amino acid residues and/or the protein with the same function lacked and ored add.It is demonstrated experimentally that can improve the resistance of plant using transcription factor provided by the invention and its encoding gene, the molecular breeding for coping with environment stress to plant has important value.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of relevant transcription factor of hybridization paper mulberry drought stress and its
Encoding gene and application.
Background technology
Arid is to influence the most important environmental factor of plant growth and crop yield.It is raw that arid has become influence agricultural
The serious problems of production improve crops and industrial crops to adverse circumstance using the drought-resistant ability of genetic engineering means Crop Improvement
Adaptability is the critical issue and significant problem of rearing new variety urgent need to resolve.In recent years, people from physiology, biochemistry, metabolism,
Ecology and heredity, evolve angularly has carried out numerous studies to the mechanism of the environment stresses such as plant responding arid, has accumulated rich
Rich data, in particular with the development of molecular biology, people can be in gene composition, expression regulation and signal transduction decile
Resistance of reverse mechanism of the plant to drought stress is recognized in sub- level, to improve the anti-stress performance of plant using genetic engineering means
New approach is opened up.Due to the complexity of plant stress-resistance character, the resistance ten of plant is improved using traditional breeding method
Divide difficulty, with the development of molecular biology, genetic engineering means open the new way of plant stress-resistance breeding, but efficiently degeneration-resistant
The principal element for being separated into limitation plant stress-resistance genetic engineering of gene.
Transcription factor (transcription factor) is to refer to and cis acting in eukaryotic gene promoter region
Specificity interaction occurs for element, assists rna plymerase ii in combination, adjusts the albumen of RNA synthesis rates.They are controlled
The coordinate expression of eucaryote normal development and physiological function gene.Development of plants is sufficiently complex process.DNA and protein
Main effect is played in this process, and the regulation and control to gene expression are realized by the interaction between them.Egg
White matter realizes diversity of organism, therefore the complexity of developmental regulation also must be with the diversity of the structure and function of protein
It is inseparable.Transcriptional control is the important mechanisms of eukaryotic gene expression regulation.Eukaryotic growth and development, Response to stress
And signal transduction be all orderly expressed due to gene regulation as a result, and such Space-time speciality of gene expression, mainly
Since transcription factor is deposited by interacting with the DNA cis elements in gene promoter and enhancer to modify change target gene
Chromatin Structure, and target base adjusted by the direct and indirect effect between transcription factor and its between transcription product
The transcript and expression of cause.Therefore, transcription factor plays a part of center adjustment in plant adverse circumstance signal transduction process, transcription because
Son is increasingly becoming the core content of plant stress-resistance mechanism study.The degeneration-resistant character of plant is the quantitative character of controlled by multiple genes.
The resistance (i.e. tolerance of the plant to arid, with high salt, low temperature and pest and disease damage) of plant is controlled by a gene, property
Shape is influenced by many genes and environment.Transcription factor can regulate and control the multiple and relevant gene of degeneration-resistant character expression, pass through
Enhance the effect of some key regulators to promote these adversity genes to play corresponding effect, the resistance of plant is made to obtain
Improve.It is anti-to improve certain with importing or improveing discrete function gene in the molecular breeding for improving plant reply environment stress
Property method compare, knock out or enhance the ability of regulation and control of a crucial transcription factor, be to improve stress resistance of plant to have an efficacious prescriptions
Method and approach.
Hybridization paper mulberry (Broussonetia kazinoki × B.papyrifera) is Institute of Botany, Chinese Academy of Sciences's profit
Through mostly for the new varieties selected after being hybridized with paper mulberry (B.papyrifera) with small paper mulberry (B.kazinoki).Hybridizing paper mulberry is
Greening, the composite multi-functional seeds with prominent resistance with material and feed dual-purpose, are collection afforestation, papermaking, sand control, feeding
Material, ecological protection are in the quick growing species of trees of one.It is fast with the speed of growth, yielding ability is strong, it is resistance to felling, it is adaptable and develop and use
The features such as mechanism reaches.
Invention content
The technical problem to be solved by the present invention is to how improve the resistance of plant.
To solve the above problems, present invention firstly provides a kind of transcription factors.
Transcription factor provided by the present invention, entitled BpDOX9, from hybridization paper mulberry (Broussonetia
Kazinoki × B.papyrifera), it is following protein a) or b) or c)：
A) amino acid sequence is protein shown in sequence 1 in sequence table；
B) fused protein that the N-terminal of protein shown in sequence 1 and/or C-terminal connection label obtain in sequence table；
C) by amino acid sequence shown in sequence in sequence table 1 by one or several amino acid residues substitution and/or
The protein with the same function lacked and ored add.
Sequence 1 wherein in sequence table can be made of 271 amino acid.
In order to which the protein in making a) is convenient for purifying, the amino terminal or carboxylic of protein shown in sequence 1
The upper label as shown in Table 1 of base end connection.
The sequence of table 1, label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being usually 5) | RRRRR |
| Poly-His | 2-10 (being usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
It is above-mentioned c) in PROTEIN B pDOX9, the substitution of one or several amino acid residues and/or missing and/or add
It adds as the substitution no more than 10 amino acid residues and/or lacks and ors add.
It is above-mentioned c) in PROTEIN B pDOX9 can be artificial synthesized, also can first synthesize its encoding gene, then carry out biological expression
It obtains.
It is above-mentioned c) in the encoding gene of PROTEIN B pDOX9 can be by will be shown in 165-980 of sequence 2
DNA sequence dna in lack the codons of one or several amino acid residues, and/or carry out one or several base-pairs missense it is prominent
Become, and/or holds the coded sequence for connecting label shown in table 1 to obtain at its 5 ' end and/or 3 '.
Protection scope of the present invention is also belonged to the relevant biomaterials of the BpDOX9.
It is provided by the present invention with the relevant biomaterials of the BpDOX9, can be following A 1) to A20) any one of：
A1 the nucleic acid molecules of the BpDOX9) 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.
Above, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules
Can be RNA, such as mRNA or hnRNA.
It is above-mentioned with the relevant biomaterials of the BpDOX9 in, A1) 1) or 2) or 3) nucleic acid molecules can be following institute
The gene shown：
1) its coded sequence is DNA molecular shown in 165-980 deoxyribonucleotides of sequence 2 in sequence table；
2) there is 75% or 75% or more homogeneity with the nucleotide sequence 1) limited, and encodes the DNA of the BpDOX9
Molecule；
1) or 2) 3) and the DNA molecular of the BpDOX9 is encoded with the nucleotide sequence hybridization that limits under strict conditions.
Wherein, sequence 2 is made of 1397 nucleotide in sequence table, and coded sequence is of sequence 2 in sequence table
165-980 nucleotide, protein shown in sequence 1 in polynucleotide.
Those of ordinary skill in the art can easily adopt by known method, for example, orthogenesis and point mutation side
Method is mutated the nucleotide sequence of the coding BpDOX9 of the present invention.Those have and the present invention point by manually modified
The nucleotide of nucleotide sequence 75% or higher homogeneity from obtained BpDOX9, as long as coding BpDOX9 and and Genes For Plant Tolerance
Inverse property is related, is the nucleotide sequence derived from the present invention and is equal to the sequence of the present invention.
Term " homogeneity " used herein refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this hair
The nucleotide sequence for the protein that amino acid sequence shown in the sequence 1 of bright polynucleotide forms has 75% or higher,
80% or higher 85% or higher 90% or higher 95% or higher homogeneity nucleotide sequence.Homogeneity can
With with the naked eye or computer software is evaluated.Using computer software, the homogeneity between two or more sequences can be used
Percentage (%) indicates, can be used for evaluating the homogeneity between correlated series.
The expression cassette includes promoter, encodes the nucleic acid molecules and terminator of the BpDOX9.The promoter can be
CaMV35S promoters.
The recombinant vector can be by the encoding gene of the BpDOX9 (i.e. shown in 165-980 of sequence 2
DNA molecular) recombinant plasmid that the plasmid that sets out obtains is inserted by the expression cassette of the encoding gene containing the BpDOX9.It is described
Double chain DNA molecule shown in sequence 5 is concretely first inserted into III Hes of Hind of carrier pCAMBIA1300 by recombinant vector
Between XbaI recognition sites, then by the encoding gene of the BpDOX9, (i.e. the sequence 2 of sequence table is from the ends 5' 165-980
Double chain DNA molecule shown in position) it is inserted into the recombinant vector obtained between Xba I and I restriction enzyme sites of Kpn.
The recombinant microorganism can be obtained by the way that the recombinant vector is imported the microorganism that sets out.
The microorganism that sets out can be yeast, bacterium, algae or fungi.The bacterium can be gram-positive bacterium or leather
Gram-negative bacteria.The gramnegative bacterium can be Agrobacterium tumefaciems (Agrobacterium tumefaciens).It is described
Agrobacterium tumefaciems (Agrobacterium tumefaciens) concretely Agrobacterium tumefaciems GV3101.
The transgenic plant cells system does not include propagating materials.The genetically modified plants are interpreted as including not only by institute
The first generation genetically modified plants that the encoding gene transformation receptor plant of BpDOX9 obtains are stated, also include its filial generation.For transgenosis
Plant can breed the gene, it is also possible to which the gene transfer is entered its of same species by traditional breeding method in the species
Its kind, particularly including in commercial variety.The genetically modified plants include seed, callus, intact plant and cell.
Applications of the BpDOX9 in regulation and control stress resistance of plant or preparation regulation and control stress resistance of plant product also belongs to this hair
Bright protection domain.
Any of the above-described relevant biomaterials of the BpDOX9 are in regulation and control stress resistance of plant or prepare regulation and control stress resistance of plant
Application in product also belongs to protection scope of the present invention.
The BpDOX9 also belongs to protection scope of the present invention as the application of transcription factor.
In order to solve the above technical problems, the present invention also provides a kind of methods for cultivating genetically modified plants.
A kind of method for cultivating genetically modified plants provided by the present invention, includes the following steps：It is imported into recipient plant
The nucleic acid molecules for encoding the BpDOX9 obtain the resistance genetically modified plants that resistance is higher than the recipient plant.
In the method for above-mentioned cultivation genetically modified plants, the nucleic acid molecules of the coding BpDOX9 can be it is following 1) or 2)
Or 3) shown in DNA molecular：
1) its coded sequence is DNA molecular shown in 165-980 deoxyribonucleotides of sequence 2 in sequence table；
2) there is 75% or 75% or more homogeneity with the nucleotide sequence 1) limited, and encodes the DNA of the BpDOX9
Molecule；
1) or 2) 3) and the DNA molecular of the BpDOX9 is encoded with the nucleotide sequence hybridization that limits under strict conditions.
Above, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules
Can be RNA, such as mRNA or hnRNA.
Wherein, sequence 2 is made of 1397 nucleotide in sequence table, and coded sequence is of sequence 2 in sequence table
165-980 nucleotide, protein shown in sequence 1 in polynucleotide.
Any of the above-described resistance concretely drought resistance.
Any of the above-described plant can be dicotyledon or monocotyledon.The dicotyledon concretely cross
Flower section plant；The crucifer can be arabidopsis.
It is demonstrated experimentally that a kind of relevant transcription factor of hybridization paper mulberry salt stress of present invention offer and its encoding gene can improve
The drought resistance of plant：Before Osmotic treatment, Columbia ecotype arabidopsis turns unloaded plant, homozygous transgenic L4 strains and pure
The growth conditions for closing transgenosis L15 strains are good；The survival rate of Columbia ecotype arabidopsis is 5% after Osmotic treatment, is turned
The survival rate of unloaded plant is 100% for the survival rate of 6%, L4 strains and L15 strains, it is seen then that with Columbia ecotype
Arabidopsis is compared, and the drought resistance of L4 strains and L15 strains dramatically increases.The result shows that using transcription provided by the invention because
Son and its encoding gene can improve the resistance of plant.
Description of the drawings
Fig. 1 is the agarose gel electrophoresis testing result for hybridizing paper mulberry seedling total serum IgE.
Fig. 2 is the agarose gel electrophoresis testing result of 3 ' RACE pcr amplification products and 5 ' RACE pcr amplification products.
Wherein, swimming lane M is TRANS2000 DNA molecular amount standards, and swimming lane 1 is 3 ' RACE pcr amplification products.
Fig. 3 is the agarose gel electrophoresis testing result of 5 ' RACE pcr amplification products.Wherein swimming lane M is TRANS2000
DNA molecular amount standard, swimming lane 1 are 5 ' RACE pcr amplification products,
Fig. 4 is the agarose gel electrophoresis testing result of PCR amplification BpDOX9 full-length cDNAs.Wherein, swimming lane M is
TRANS2000 DNA molecular amount standards, swimming lane 1 are pcr amplification product.
Fig. 5 is relative expression quantity of BpDOX9 genes under the conditions of Different stress.
Fig. 6 is the subcellular localization result of BpDOX9 genes.
Wherein, A-D is the knot of recombinant expression carrier pCAMBIA1302-BpDOX9 transformation of tobacco epidermal cell transient expressions
Fruit, E-H are the result of carrier pCAMBIA1302 transformation of tobacco epidermal cell transient expressions；A and E is that the epidermal tobacco of conversion is thin
Born of the same parents under blue channel (observation DAPI, confirm the position of nucleus) form, B and F be the Tobacco Epidermis that converts glimmering
The form of (observation of GFP fluorescins) under optical channel, C and G are the form under bright field, D and the superposition that H is three visual fields.
Fig. 7 is that the transcriptional activation activity of BpDOX9 genes is analyzed.
Wherein, A is position of the transgenic yeast on tablet；B is transgenic yeast in the SD cultures without His and Trp
Upgrowth situation on base；C is that the betagalactosidase activity of transgenic yeast detects.
Specific implementation mode
The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
TRANS2000 DNA molecular amount standards are Beijing Quanshijin Biotechnology Co., Ltd's product.
SD culture mediums without His and Trp are the general Jino Science and Technology Ltd. product in Beijing, product identification YGM003A-
17。
Carrier PMD-18T is Takara Products, catalog number D103A.
Yeast expression carrier pBridge containing GAL4 binding domain is purchased from Clontech companies of the U.S., article No. 630404.
Yeast AH109 strains containing His3 and LacZ reporters are purchased from Clontech companies of the U.S., and cat. no is
K1612-1。
Carrier pCAMBIA1302 and Agrobacterium EHA105 is the production of prosperity biotechnology Co., Ltd of Beijing ancient cooking vessel state
Product, catalog number are respectively MCV034-N and MCC028.
Tobacco bred Nicotiana tabacum cv Xanth are recorded in the following literature：Hoi PX,Quy TD,
Nghia PT,Tuteja N:Transfer of gene encoding for DNA unwinding helicase(pdh45)
into tobacco plants(Nicotiana tabacum L.cv Xanthi)by using Agrobacterium and
Analysis of the Transformed plants.TAP CHI SINH HOC 2015,25(3):83-92..Below
Middle tobacco bred Nicotiana tabacum cv Xanth are referred to as tobacco.
Hybridization paper mulberry (Broussonetia kazinoki × B.papyrifera) is Institute of Botany, Chinese Academy of Sciences's profit
It, can be from Beijing Qiao Na through the kind educated of more being commissioned to train after being hybridized with paper mulberry (B.papyrifera) with small paper mulberry (B.kazinoki)
Gloomy development in science and technology Co., Ltd purchase.
Recombinant vector pBridge-JcERF is recorded in the following literature：M.Tang,J.Sun,Y.Liu,F.Chen,
S.Shen,Isolation and functional characterization of the JcERF gene,a putative
AP2/EREBP domain-containing transcription factor,in the woody oil plant
Jatropha curcas,Plant Mol.Biol.63(2007)419–428.
Embodiment 1, the acquisition for hybridizing the relevant transcription factor encoding gene of paper mulberry drought stress
One, the clone of the 3 ' terminal sequences of the relevant transcription factor encoding gene BpDOX9 of hybridization paper mulberry drought stress
1, vegetable material processing and the extraction of total serum IgE
Arid (20%, PEG6000) processing is carried out to the seedling of 8 weeks hybridization paper mulberries of normal growth, processing carries after 6 hours
The total serum IgE of hybridization paper mulberry seedling is taken, 1% agarose gel electrophoresis detection is carried out.
The results are shown in Figure 1, the results showed that, the total serum IgE of extraction has two apparent electrophoretic bands, is followed successively by from top to bottom
28S RNA and 18S RNA, show to obtain that purity is higher, more complete total serum IgE.
2, the clone of the 3 ' terminal sequences of the relevant transcription factor encoding gene BpDOX9 of hybridization paper mulberry drought stress
(1) specifically drawn according to conservative region primers according to published plant sequence search conservative region
Object sequence is as follows：F1：5′-TGGTTCTTTACTTGGTTAGCCG-3′.
(2) total serum IgE for the hybridization paper mulberry seedling extracted using above-mentioned steps 1 uses PrimeScript as templateTM 1st
Strand cDNA Synthesized Kit kits (Takara Products) and with reference to the requirement of kit specification, instead
Turn to synthesize its first chain cDNA.Reaction system and reaction condition are as follows：1 μ L, Total RNA of Oligo-dT (10pmol/ μ l) (≤
1 μ g) 2 μ L, dNTP Mixture (10mmol/L each) 1.0 μ L, 5 × Buffer, 4.0 μ L, RNase Inhibitor (40U/
μ L) 0.5 μ L, PrimeScript RTase (200U/ μ L) 0.5 μ l, RNase-free distilled water, 11 μ L；65℃
5min, 42 DEG C of 45min, 70 DEG C of 15min.By the first chain cDNA of synthesis be stored in -20 DEG C it is spare.
(3) the first chain cDNA obtained again using step (2) is template, using primers F 1 and primer OligodT-adaptor
5 '-GATTTCTGTCCGACGACTTTTTTTTTTTTTTTTTT-3 ' carry out PCR amplifications obtain 3 ' RACE pcr amplification products.
PCR reaction systems are：CDNA templates, F1 primers and OligodT-adaptor each 1 μ L, 10 × Buffer 2.5 μ L, dNTP
2 μ L of Mixture (10mmol/L each), Taq enzyme 0.25 μ L and ddH2O 12.25μL.Response procedures are：94 DEG C of pre-degenerations
5min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 60s, totally 36 recycle；72 DEG C of extension 10min.
(4) after reaction, 1% agarose gel electrophoresis detection is carried out to 3 ' RACE pcr amplification products, as a result such as Fig. 2
It is shown, wherein swimming lane 1 is 3 ' RACE pcr amplification products.The result shows that obtaining length through 3 ' RACE PCR amplifications and being about
The target fragment of 850bp.
(5) it recycles and purifies 3 ' RACE pcr amplification products, be connected on carrier PMD-18T, connection product conversion
Bacillus coli DH 5 alpha competent cell, screening positive clone carry out bacterium solution PCR identifications, and the plasmid for extracting positive colony is surveyed
Sequence, and BLAST analyses are carried out to sequencing result.The result shows that the length of the segment is 857bp, deoxyribonucleotide sequence
Row are as shown in sequence 3 in sequence table.
Two, the clone of the 5 ' terminal sequences of the relevant transcription factor encoding gene BpDOX9 of hybridization paper mulberry drought stress
(1) cDNA sequence design primer is held according to the BpDOX9 genes 3 ' that above-mentioned steps one obtain：R1：5′-
GAGGAGGAAGATGGAACCG-3′。
(2) it using the total serum IgE of the hybridization paper mulberry seedling of step 1 extraction as template, is tried using 5 ' RACE of Promega companies
Agent box and with reference to kit specification, reverse transcription synthesizes its first chain cDNA.
Reaction system and condition are as follows：1 μ L RNA, 1 μ L 5'-CDS primer A, 1 μ L SMART II A oligo, 1
μ L DTT (20mM), 1 μ L dNTP Mix (10mM), 1 μ L MMLV Reverse Transcriptase, 2 μ L 5X First-
Strand Buffer and 2 μ L sterile H2O；70 DEG C of 2min, on ice 2min, 42 DEG C of 1.5h, 72 DEG C of 7min.
(3) the first chain cDNA obtained using step (2) is template, and using primer R1 and primer UPM, (Promega companies produce
Product：Long(0.4μM)：5'-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3', Short (2 μ
M)：5'-CTAATACGACTCACTATAGGGC-3') pairing carries out PCR amplification, obtains 5 ' RACE pcr amplification products.PCR is anti-
The system is answered to be：1 μ L 50X Advantage 2 Polymerase Mix, 34.5 μ L PCR-Grade Water, 5 μ L 10X
2 PCR 2 Polymerase Mix of Buffer, 1 μ L dNTP Mix (10mM), 1 μ L 50X Advantage of Advantage,
5 μ L UPM, 1 μ L primers R, 2.5 μ L cDNA templates.Reaction condition is：94℃30s；68 DEG C of 30s, 70 DEG C of 60s, totally 40 are followed
Ring；70 DEG C of extension 10min.
(4) after reaction, 1% agarose gel electrophoresis detection is carried out to 5 ' RACE pcr amplification products, as a result such as Fig. 3
It is shown, wherein swimming lane 1 is 5 ' RACE pcr amplification products.The result shows that obtaining length through 5 ' RACE PCR amplifications and being about
The target fragment of 750bp.
(5) it recycles and purifies 5 ' RACE pcr amplification products, be connected on PMD-18T carriers, connection product conversion
Bacillus coli DH 5 alpha competent cell, screening positive clone carry out bacterium solution PCR identifications, and the plasmid for extracting positive colony is surveyed
Sequence carries out BLAST analyses to sequencing result.The result shows that the length of the segment is 779bp, deoxyribonucleotide sequence
As shown in sequence 4 in sequence table.
Three, hybridization the relevant transcription factor encoding gene BpDOX9 full length cDNA sequences of paper mulberry drought stress acquisition and
PCR is detected
1, hybridize the acquisition of the relevant transcription factor encoding gene BpDOX9 full length cDNA sequences of paper mulberry drought stress
Overlay region of the length obtained using above-mentioned steps one and step 2 between 857bp and 779bp segments, by
The full length cDNA sequence that Contig softwares splice, in deoxyribonucleotide sequence such as sequence table shown in sequence 2, by sequence
Unnamed gene shown in row 2 is BpDOX9, is ORF from 5 ' end 165-980, encodes the egg being made of 271 amino acid residues
White matter, the albumen are named as BpDOX9, and the amino acid sequence of the albumen is the sequence 1 in sequence table.
2, PCR is detected
(1) following primer is designed according to BpDOX9 full length gene cDNA sequences：
F2：5′-GTTTGTCTCTTTCTTTCCAACCTTTTTCCT-3′；
R2：5′-GGTTCATCCATTCATATCCAATCATAA-3′.
(2) the first chain cDNA synthesized through reverse transcription using the total serum IgE of the hybridization paper mulberry seedling of above-mentioned step 1 extraction is mould
Plate carries out PCR amplification using F2 and R2, obtains pcr amplification product.
(3) 1% agarose gel electrophoresis detection is carried out to pcr amplification product, the results are shown in Figure 4.The result shows that warp
PCR amplification obtains the segment that length is about 1450bp.
(4) it recycles and purifies the product, be connected on PMD-18T carriers, connection product converts bacillus coli DH 5 alpha
Competent cell, screening positive clone carry out bacterium solution PCR identifications, and the plasmid for extracting positive colony is sequenced.
Sequencing result shows that the pcr amplification product has nucleotide sequence shown in sequence 2 in sequence table.
The expression pattern analysis of embodiment 2, BpDOX9 genes under the conditions of the different abiotic stress factors
Low temperature, with high salt, arid and methyl jasmonate treatment are carried out for analyzing hybridization paper mulberry BpDOX9 bases to hybridization paper mulberry
Because of the expression under abiotic stress.In the medium by the seed kind for hybridizing paper mulberry, after growing to 8 weeks, seedling is divided
It carry out not low temperature (4 DEG C), (200mM NaCl solutions) with high salt, arid (PEG6000, 20%) and abscisic acid (ABA, 10%) processing.
In the different disposal period, seedling is collected respectively, for extracting RNA.It is described that the specific method is as follows：
Low-temperature treatment：Hybridization paper mulberry seedling is placed in 4 DEG C of incubators, respectively in illumination cultivation 0h, 1h, 3h, 6h and 12h
It is sampled after hour.
High salt treatment：By hybridize paper mulberry seedling root system be placed in 200mM NaCl solutions, respectively illumination cultivation 0h,
It is sampled after 1h, 3h, 6h and 12h hours.
Osmotic treatment：The root system for hybridizing paper mulberry seedling is placed in 20%PEG6000In solution, respectively illumination cultivation 0h,
It is sampled after 1h, 3h, 6h and 12h hours.
Abscisic acid processing：By hybridize paper mulberry seedling root system be placed in 10%ABA solution, respectively illumination cultivation 0h, 1h,
It is sampled after 3h, 6h and 12h hours.
The total serum IgE for extracting above-mentioned processing hybridization paper mulberry seedling respectively, then uses PrimeScriptTM 1st Strand
CDNA Synthesized Kit kits (Takara Products) and with reference to the requirement of kit specification, reversion synthesis its
First chain cDNA.Then expression of quantifying PCR method analysis BpDOX9 genes under the conditions of the different abiotic stress factors is used
Pattern.
It is as follows：
1, the design of primer
Its specific primer QF and QR is designed according to the cDNA sequence of hybridization paper mulberry BpDOX9, and is made with Bpactin genes
For the internal reference of reaction, primer sequence is as follows：
QF：5′-ATGCGGAAAACTACAGCATGAC-3′；
QR：5′-CAAAACCATGACATTCTACTCCCTG-3′；
Bpactin-F：5′-CCGTGCTCAATGGGATACTTC-3′；
Bpactin-R：5′-CCCTCGTCTGTGACAATGGTAC-3′.
2, quantitative PCR
The cDNA for hybridizing paper mulberry seedling using above-mentioned processing respectively carries out Q- as template using the primer that above-mentioned steps 1 design
PCR amplification.Reaction system is：SYBR Green Mix 10 μ L, QF 0.4 μ L, QR0.4 μ L, ddH2O7.2 μ L and cDNA template 2
μ L (are used as template) after reverse transcription product is diluted 10 times, total volume is 20 μ L.Real-time quantitative PCR reaction is complete using two-step method
At response procedures are：95℃60s；95 DEG C of 15s, 65 DEG C of 45s；40 cycles.The analysis Mx3000p of the data obtained and Ct values
Software carries out.
The results are shown in Figure 5.The result shows that the transcriptional level of BpDOX9 genes is obviously induced by drought stress, with
The relative expression quantity of the extension of stress time, BpDOX9 genes increases sharply, and reaches within 3 hours maximum value to handling, after 12h
Expression quantity continuously decreases；The expression quantity variation of BpDOX9 genes is slow under high-salt stress and the acid stress that falls off；In low-temperature treatment
Under, the expression quantity of BpDOX9 genes changes unobvious.
The functional verification of embodiment 3, BpDOX9 transcription factors
One, the subcellular localization of BpDOX9
1, the NcoI of carrier pCAMBIA1302 is identified that the DNA small fragments between sequence and SpeI identification sequences replace with core
Nucleotide sequence is the double chain DNA molecule shown in the ends 5' the 165th to the 980th of sequence 2 of sequence table, is recombinantly expressed
Carrier is named as pCAMBIA1302-BpDOX9.
2, the above-mentioned recombinant expression carrier pCAMBIA1302-BpDOX9 of 5 μ g are transferred in Agrobacterium EHA105, obtain weight
Group Agrobacterium.
3, by recombinational agrobacterium transformation of tobacco epidermal cell (A-D in Fig. 6), while to be transferred to empty carrier pCAMBIA1302
Tobacco Epidermis as a contrast (E-H in Fig. 6).
4, the Agrobacterium for completing step 3 is cultivated 24-48 hours, and 10~20min is then dyed in DAPI (10mM), is swashing
It observes and takes a picture under light confocal scanning microscope (Bio-Rad MRC 1024).
As a result see Fig. 6.The result shows that being transferred to the albumen distribution expressed in the transgenic cell of empty carrier pCAMBIA1302
Entire intracellular, such as H in F and Fig. 6 in Fig. 6；And the transgenosis for being transferred to recombinant expression carrier pCAMBIA1302-BpDOX9 is thin
The albumen expressed in born of the same parents is then positioned in nucleus, such as D in B and Fig. 6 in Fig. 6.The result shows that：BpDOX9 is positioned at nucleus
It is interior.
Two, the transcriptional activation activity analysis of BpDOX9
1, the BamHI of the Yeast expression carrier pBridge containing GAL4 binding domain is identified that sequence and SalI identify sequence
Between DNA small fragments to replace with the sequence 2 that nucleotide sequence is sequence table double shown in the ends 5' the 165th to the 980th
Ssdna molecule keeps the other sequences of Yeast expression carrier pBridge constant, obtains recombinant expression carrier, be named as
pBridge-BpDOX9。
2, recombinant vector pBridge-BpDOX9 is imported into the yeast AH109 strains containing His3 and LacZ reporters
In, obtain the transgenic yeast containing recombinant vector pBridge-BpDOX9；
Carrier pBridge is imported into the yeast AH109 strains containing His3 and LacZ reporters, is contained
The transgenic yeast of pBridge, as negative control；
Recombinant vector pBridge-JcERF is imported into the yeast AH109 strains containing His3 and LacZ reporters, is obtained
To the transgenic yeast containing pBridge-JcERF, as positive control.
3, by the transgenic yeast containing recombinant vector pBridge-BpDOX9 that above-mentioned steps 2 obtain, contain pBridge
Transgenic yeast and transgenic yeast containing pBridge-JcERF respectively in the SD culture mediums (SD/- without His and Trp
His-Trp it is cultivated on).
The results are shown in Figure 7.The result shows that the transgenic yeast containing pBridge is cultivated in the SD without His and Trp
It cannot be grown on base, and the transgenic yeast containing recombinant vector pBridge-BpDOX9 and turn containing pBridge-JcERF
Gene yeast can be grown on the SD culture mediums without His and Trp and aobvious blue.Illustrate that BpDOX9 lives with transcriptional activation
Property.
The acquisition of embodiment 4, genetically modified plants
One, the structure of recombinant plasmid
With carrier pCAMBIA1300 (CAMBIA Products) for skeleton carrier, in Hind III and XbaI enzyme cutting site
Between insetion sequence table sequence 5 shown in double chain DNA molecule (CaMV35S promoters), between I restriction enzyme site of Xba I and Kpn
The sequence 2 of insetion sequence table double chain DNA molecule shown in the ends 5' the 165th to the 980th, obtains recombinant plasmid first.
With carrier pCAMBIA1300 (CAMBIA Products) for skeleton carrier, in Hind III and XbaI enzyme cutting site
Between insetion sequence table sequence 5 shown in double chain DNA molecule (CaMV35S promoters), obtain recombinant plasmid second.
Two, the acquisition of transfer-gen plant
1, the recombinant plasmid first for obtaining step 1 imports Agrobacterium tumefaciems GV3101, obtains recombinational agrobacterium.
2, the recombinational agrobacterium for taking step 1 to obtain transfects Columbia ecotype arabidopsis by flower-dipping method, then cultivates
Plant simultaneously harvests seed, as T1For seed.
3, the seed that step 2 obtains is seeded in the MS solid mediums of the hygromycin containing 300mg/L, screen resistant plant
(T1For plant).
4, T is extracted1For the genomic DNA of plant, PCR identifications are carried out.
The primer pair of PCR identifications is as follows：
QF：5′-ATGCGGAAAACTACAGCATGAC-3′；
QR：5′-CAAAACCATGACATTCTACTCCCTG-3′；
If PCR is accredited as positive (PCR amplification obtains the amplified production of about 280bp), which is transfer-gen plant.
The offspring of each plant is a strain.
5, PCR in step 4 is accredited as positive plant selfing and harvests seed (T2For seed).
6, the cultivating seeds for obtaining step 5 are plant (T2For plant) and single plant sowing (T3For seed).
7, by T3It is seeded into the MS solid mediums of the hygromycin containing 300mg/L for seed, screens the unseparated homozygosis of resistance
Single plant (screens its T3The T of Resistant segregation does not occur for seed2For plant, which is the transfer-gen plant of homozygosis), by its kind
Son (T3For seed) carry out step 3 every detection and identification.
Three, turn the acquisition of empty carrier plant
It replaces recombinant plasmid first to carry out step 2 with recombinant plasmid second, obtains turning empty carrier plant.
Four, drought resistance
Seed to be measured is as follows：L4 strains (100 T3For seed), L15 strains (100 T3For seed), turn empty carrier plant
(100 T3For seed) and Columbia ecotype arabidopsis (100 seeds).L4 strains and L15 strains are two taken at random
The transgenic line of a homozygosis.
Seed to be measured is sowed in the flowerpot equipped with identical weight soil and is cultivated, and the timing since sowing, the 1st to 14 day just
Often management, the 15th to 25 day Osmotic treatment (Osmotic treatment continues not water), start within the 26th day normal management (restore watering,
Abbreviation rehydration).Survival rate is counted at the end of 28th day (after rehydration 3 days).
The survival rate of Columbia ecotype arabidopsis is 5%, and the survival rate for turning unloaded plant is depositing for 6%, L4 strains
Motility rate is that the survival rate of 100%, L15 strains is 100%.The result shows that compared with Columbia ecotype arabidopsis, L4 strains
It is dramatically increased with the drought resistance of L15 strains.
Claims (8)
1. a kind of protein is protein shown in sequence 1 in sequence table.
Any one of 2. it is following A 1 with the relevant biomaterial of protein described in claim 1) 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) nucleic acid molecules are sequence in sequence table
DNA molecular shown in 165-980 deoxyribonucleotides of row 2.
4. the application of protein described in claim 1 is following a1) or a2)：
A1) the application in regulating and controlling stress resistance of plant；
A2) the application in preparing stress resistance of plant product；
The plant is arabidopsis or hybridization paper mulberry；
The resistance is drought resistance.
5. the application of relevant biological material described in Claims 2 or 3 is following b1) or b2)：
B1) the application in regulating and controlling stress resistance of plant；
B2) the application in preparing stress resistance of plant product；
The plant is arabidopsis or hybridization paper mulberry；
The resistance is drought resistance.
6. application of the protein described in claim 1 as transcription factor.
7. a kind of method for cultivating genetically modified plants, includes the following steps：It is imported into recipient plant described in coding claim 1
The nucleic acid molecules of protein obtain the resistance genetically modified plants that resistance is higher than the recipient plant；
The plant is arabidopsis or hybridization paper mulberry；
The resistance is drought resistance.
8. according to the method described in claim 7, it is characterized in that：The nucleic acid molecules of protein are described in coding claim 1
DNA molecular shown in 165-980 deoxyribonucleotides of sequence 2 in sequence table.
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Citations (2)
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| CN1796559A (en) * | 2004-12-21 | 2006-07-05 | 华中农业大学 | Using gene of transcriptional factor OSNACX of paddy to increase drought resistance and salt tolerant abilities of plants |
| CN101045929A (en) * | 2007-03-12 | 2007-10-03 | 华中农业大学 | Raising plant cold endurance and salt tolerance by means of transcription factor gene SNAC2 of rice |
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|---|---|---|---|---|
| CN1796559A (en) * | 2004-12-21 | 2006-07-05 | 华中农业大学 | Using gene of transcriptional factor OSNACX of paddy to increase drought resistance and salt tolerant abilities of plants |
| CN101045929A (en) * | 2007-03-12 | 2007-10-03 | 华中农业大学 | Raising plant cold endurance and salt tolerance by means of transcription factor gene SNAC2 of rice |
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