CN102168094B - Kandelia candel (L.) druce ERF (ethylene-responsive element binding factor) transcription factor as well as encoding gene, expression vector and application thereof - Google Patents
Kandelia candel (L.) druce ERF (ethylene-responsive element binding factor) transcription factor as well as encoding gene, expression vector and application thereof Download PDFInfo
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Abstract
The invention discloses a kandelia candel (L.) druce ERF (ethylene-responsive element binding factor) transcription factor as well as an encoding gene, an expression vector and application thereof. In the invention, the nucleic acid sequence of the ERF transcription factor cDNA separated from mangrove plant kandelia candel (L.) druce is as shown in SEQ ID No.1. After the ERF transcription factor cDNA is transformed to tobacco, the contents of osmoregulation substances (such as proline) of a transgenic plant are obviously increased, and the salt tolerance of the transgenic plant is obviously improved. An experimental result indicates that the ERF transcription factor can response an adverse signal, thereby effectively improving environmental stress resistances of the plant such as salt and base resistance, diseases and pets resistance and the like.
Description
Technical field
The present invention relates to a kind of transcription factor cDNA relevant to stress resistance of plant and proteins encoded thereof, a cDNA sequence that relates in particular to from mangrove plant autumn eggplant [Kandelia candel (L.) Druce] institute's coding ERF transcription factor of separating, cloning, the invention still further relates to the high efficiency plant expression vector and their application in improving stress resistance of plant that contain this cDNA sequence, belong to the transcription factor field with the anti-retrocorrelation of plant.
Background technology
The adverse circumstance of plant comprises abiotic stress (as arid, saline and alkaline and low temperature etc.) and biological adverse circumstance (disease, insect pest and the weeds that cause as fungi, bacterium, virus and nematode etc. endanger).Arid, saline and alkaline and low temperature is three kinds of main abiotic stress factors that affect terrestrial plant growth and restriction crop yield; Wherein, very large proportion is occupied to the impact of crop yield in the saltings.According to statistics, saltings, the world accounts for 7.6% of Global land area, along with process of industrialization is accelerated, the soil salinization area of China constantly enlarges, the salinization soil area approximately has 2,600 ten thousand hectares at present, and wherein Saline approximately has 6,700,000 hectares, has seriously restricted the utilization of agricultural to the soil.One of serious adverse circumstance factor as often occuring and endanger in low temperature, and it not only affects the seasonality growth of plant, and the geographic that also affects plant distributes.The abiotic stress of saline and alkaline formation can cause osmotic stress to plant, causes the environment seepage gesture to cause the vegetable cell dehydration lower than the vegetable cell osmotic potential, and serious caused cell turgor completely loses, and even causes plant dead.
Disease and pest is the important biomolecule stress factors that causes crop production reduction, forest harm.The annual forest disease and pest generation area of China is about 1.2 hundred million mu of left and right, and the great disease and pest of national farm crop in 2010 still for laying particular stress on, occurs the time, and overall degree slightly overweights last year, area occurs be about 7,000,000,000 mu (inferior), totally and remain basically stable last year.All there is serious sick worm harm in the Important Economic crops such as paddy rice, corn, cotton, cause huge financial loss.Plant has obtained complicated plant defense (plant defense responses) mechanism in the process that the adaptation pathogen infects.After plant identification pathogen, by a series of signal transmittance process, finally induce the expression of relevant plant defense gene.The transcriptional control of plant defense gene (transcription regulation) has become one of field most active in plant defense research.Yet the overexpression of single plant defense gene only can improve plant to the resistivity of certain pathogen, and the overexpression of a transcription factor can activate the expression of a plurality of downstreams disease-resistant gene.Therefore, utilizing transcription factor to improve the comprehensive disease resistance of plant, is a kind of very potential method.
The plant stress-resistance genetically engineered is to import the discrete function gene to improve certain resistance, does not obtain purpose comprehensive, the essence improvement but reach the resistance that makes plant.The resistance of plant is not that the expression by single or a few gene embodies, but by the quantitative character of controlled by multiple genes.Although clone successively a large amount of anti contravariance related genes at present from each kind of plant, these gene great majority can only increase certain single resistance of plant, can not comprehensively improve on the whole the resistance of plant.The by-pass cock that transcription factor is expressed as functional gene, can regulate accurately different genes, bringing into play crucial effect in plant adverse circumstance signal transduction process, so by strengthening the effect of certain transcription factor, can impel a plurality of and degeneration-resistant relevant functional gene to express, this is to make the plant stress-resistance proterties obtain a very effective approach of comprehensive improvement.
The AP2/EREBP proteinoid is the peculiar class transcription factor of higher plant, ERF (ethylene-responsive element binding factor) class transcription factor is a subfamily of AP2/EREBP transcription factor family, and each member is contained a very conservative DNA who is comprised of about 60 amino acid in conjunction with the territory.From structural analysis of protein, ERF class transcription factor contains 4 functional domains, be that DNA is in conjunction with territory (DNA-binding domain), transcriptional regulatory domain (transcription regulation domain) (comprise Activation and inhibition territory), oligomerization site (oligomerization) and nuclear localization signal (nuclear localization signal, NLS).Wherein, the ERF/AP2DNA binding domains of high conservative contains and comprises 3 β-pleated sheet structures and 1 α spiral by what 60～70 amino-acid residues formed, by with goal gene upstream promoter zone in be rich in GC cis-acting elements (as DRE element, GCC-box etc.) interact, participate in the regulating plant cell cycle, grow, cell proliferation, secondary metabolism, biology and abiotic stress is replied and the multi-signal approach such as plant hormone signal conduction, play an important role in the stress signal transmittance process.The ERF transcription factor can regulate and control a plurality of expression of resisting the relevant functional gene such as arid, high salt, low temperature and pathogenic bacteria with plant, strengthen on the whole the resistance of plant, improve its stability, resistance of reverse for genetically engineered improvement plant has huge potential using value, and huge application prospect will be arranged.
Studies show that, the expression of most of ERF gene all is subjected to inducing of the environment stresses such as arid, saline and alkaline, low temperature, freeze injury, disease and mechanical wounding, clone new ERF class transcription factor gene, study its basic biological characteristics and function, can be whole plant stress-resistance gene regulatory network and stress response reaction mechanism and provide fundamental basis, and provide certain basic substance for Crop Improvement resistance.
Autumn eggplant [Kandelia candel (L.) Druce] is one of typical halophytes one mangrove forest seeds, and leaf is to life, and fruit is elongated, the common high 1.5-6 rice of root of Kandelia layer.In autumn eggplant growing environment, the soil matrix salinity is high, is subjected to the periodicity dipping time of high sea brine long, and the cover of evolving out in long-term evolutionary process is different from the Mechanisms of Salt Resistance of terrestrial plant or freshwater plant.Autumn, eggplant had extremely strong Salt And Alkali Tolerance ability; not only play an important role in safeguarding coastal ecology balance, environmental monitoring, water body purification; main is to have formed its distinctive gene to consist of and accurate expression regulation mode due to this halophytes in the long term growth evolutionary process, can synthesize and keep more salt tolerant Osmotic Stress Tolerance Protective substances.Therefore, the transcription factor gene that the clone is closely related with resistance from Kandelia candel mangrove can not only provide the excellent genes source for genetic engineering breeding, and seem particularly important and urgent for the crop breeding for stress tolerance.
Summary of the invention
One of the object of the invention is to provide a kind of ERF transcription factor cDNA and proteins encoded thereof that is closely related with resistance that separate, clone from mangrove plant autumn eggplant [Kandelia candel (L.) Druce].
Two of the object of the invention is to provide the expression vector that contains above-mentioned ERF transcription factor cDNA and the host cell that contains this expression vector.
Three of the object of the invention is described ERF transcription factor cDNA and proteins encoded thereof to be applied to improve or the stress resistance of regulating plant.
Above-mentioned purpose of the present invention is achieved through the following technical solutions:
A kind of ERF transcription factor cDNA that separates from autumn eggplant [Kandelia candel (L.) Druce], its Nucleotide be (a), (b) or (c) shown in:
(a), the Nucleotide shown in SEQ ID No.1;
(b), amino acid whose Nucleotide shown in coding SEQ ID No.2;
(c), with the Nucleotide that the complementary sequence of SEQ ID NO:1 can be hybridized in rigorous hybridization conditions, the amino acid of the coded protein of this Nucleotide is shown in SEQ ID NO:2.
Preferably, the ERF transcription factor cDNA that separates from the autumn eggplant of the present invention is the Nucleotide shown in SEQ ID No.1.
Two of the object of the invention be to provide by above-mentioned transcription factor cDNA coded can improve plant to the ERF transcription factor of tolerance to environmental stress, its amino acid be (a) or (b) shown in:
(a), the amino acid shown in SEQ ID No.2;
(b), the replacement by one or more amino-acid residues with the amino acid shown in SEQ ID No.2, disappearance are or/and insert and derive the protein variant that still has ERF functional transcription factor shown in SEQ ID No.2 or activity that obtains;
Preferably, ERF transcription factor of the present invention is the amino acid shown in SEQ ID No.2.
Described " a plurality of " mean 2-8 usually, are preferably 2-4, and this depends on position or the amino acid whose kind of amino-acid residue in ERF transcription factor three-dimensional structure; Described " replacement " refers to replace one or more amino-acid residues with different amino-acid residues respectively; Described " disappearance " refers to the minimizing of amino-acid residue quantity, that is to say to lack respectively one or more amino-acid residue; Described " insertion " refers to the change of amino acid residue sequence, relative natural molecule, and described change causes adding one or more amino-acid residues.
Protein variant of the present invention can be produced by genetic polymorphism or manual operation, and these working method are generally this area and understand.For example, can prepare by the sudden change of DNA aminoacid sequence variant or the fragment of ERF transcription factor, the method for wherein said mutagenesis or change polynucleotide is known by this area.Conservative replacement is that a kind of amino-acid residue is replaced to the another kind of amino acid with similar quality.
ERF transcription factor of the present invention and encoding gene thereof comprise naturally occurring sequence and two kinds of forms of variant." variant " means substantially similar sequence, and for polynucleotide, variant comprises the disappearance, insertion of the one or more Nucleotide of one or more site in natural polynucleotide or/and replace.For polynucleotide, conservative variant comprises due to the degeneracy of genetic code those variants of the aminoacid sequence that does not change coding.Naturally occurring variant like that can be identified by existing Protocols in Molecular Biology.The variant polynucleotide also comprise the polynucleotide in synthetic source, the amino acid whose polynucleotide variant shown in SEQ ID No.2 of still encoding that for example adopts site-directed mutagenesis or obtain by the method for recombinating.Those skilled in the art can screen or estimate by following molecular biotechnology means function or the activity of the coded albumen of variant polynucleotide: DNA binding activity, interaction between albumen, the effect of expressing in the activation situation of genetic expression in instantaneous research or transgenic plant etc.
The invention provides and a kind ofly improve plant to the method for tolerance to environmental stress, comprising: the ERF transcription factor cDNA that the present invention is separated is incorporated in plant or vegetable cell, can effectively improve target plant to the resistance of environment-stress; For example.ERF transcription factor cDNA of the present invention can be incorporated in the target plant cell, cultivate and screen the transgenic plant that obtain the resistance raising of environment-stress, wherein, described environment-stress comprises abiotic stress (arid, low temperature, saline and alkaline etc.) or biological adverse circumstance (disease and pest that causes as fungi, bacterium, virus and nematode etc. and weeds harm etc.).
The present invention also provides the plant expression vector that contains described ERF transcription factor gene and the host cell that contains this expression vector.
Be connected with expression regulation element ERF transcription factor cDNA of the present invention is exercisable, obtain to express the plant expression vector of this gene in plant." exercisable connection " refers to functional connection between two or more elements, and the element of exercisable connection can be adjacency or non-adjacent.This plant expression vector can be by 5 ' end non-coding region, and the Nucleotide shown in SEQ ID No.1 and 3 ' non-coding region form, and wherein, described 5 ' end non-coding region can comprise that promoter sequence, enhancer sequence are or/and the translation enhancement sequences; Described promotor can be composition promotor, inducible promoter, tissue or organ specific promoters; Described 3 ' non-coding region can comprise terminator sequence, mRNA cutting sequence etc.Suitable terminator sequence can be taken from the Ti-plasmid of agrobacterium tumefaciens, for example octopine synthetic enzyme and rouge alkali synthetase terminator.In addition, those skilled in the art can be optimized the Nucleotide shown in SEQ ID No.1 to strengthen the expression in plant.For example.Can adopt the preference codon of target plant to be optimized synthetic polyribonucleotides to strengthen the expression in target plant.
Plant expression vector of the present invention also can contain the selected marker who is useful on the selection transformant.The selected marker is used for the cell or tissue of selection through transforming.Marker gene comprises: the gene of coding antibiotics resistance and the gene etc. of giving the herbicidal compound resistance.In addition, described marker gene also comprises phenotypic markers, such as beta-galactosidase enzymes and fluorescin etc.
The invention still further relates to described ERF transcription factor cDNA is incorporated in plant to improve the stress resistance of plant; Described " introducing " refers to that the ERF transcription factor cDNA is imported to the inner such mode of vegetable cell to be transformed into polynucleotide or polypeptide in plant.The method that described polynucleotide or polypeptide are incorporated in plant is known by this area, include but not limited to stable conversion method, instantaneous conversion method and virus-mediated method etc." stable conversion " refers to that the polynucleotide constructs that is introduced into is integrated in the genome of vegetable cell and can be hereditary by its filial generation; " instantaneous conversion " refers to that polynucleotide are introduced in plant but can only transient expression or existence in plant.
Described conversion scheme and with visual plant (monocotyledons or dicotyledons) or the type of vegetable cell and changing for transforming of the scheme of described polynucleotide or polypeptide introduced plant.The appropriate method of described polynucleotide or polypeptide being introduced vegetable cell comprises: microinjection, electroporation, agriculture bacillus mediated conversion, direct gene transfer and bombardment of high speed trajectory etc.In specific embodiment, can utilize multiple instantaneous conversion method that ERF transcription factor cDNA of the present invention is offered plant.In other embodiments, ERF transcription factor cDNA of the present invention can be by contacting be incorporated in plant with virus or viral nucleic acid plant, usually, such method relates in ERF transcription factor cDNA construct introducing viral DNA of the present invention or RNA molecule.
The cell regeneration stable conversion plant (McCormick et al.Plant Cell Reports.1986.5:81-84) that utilizes ordinary method to make to have transformed.The present invention can be used for transforming any floristics, includes but not limited to: monocotyledons or dicotyledons.Preferred, described target plant comprises farm crop, vegetables or ornamental plant, fruit tree etc., for example, can be corn, paddy rice, Chinese sorghum, wheat, soybean, potato, barley, tomato, Kidney bean, peanut, sugarcane or cotton etc.
After being transformed into ERF transcription factor cDNA of the present invention in tobacco, the Osmotic Adjustment Substances such as the proline(Pro) of transfer-gen plant have obvious rising, and the salt tolerance of transfer-gen plant is significantly improved.Experimental result shows, ERF transcription factor cDNA of the present invention can effectively improve the tolerance to environmental stress such as the Salt And Alkali Tolerance, disease and insect resistance of plant.
The term definition that arrives involved in the present invention
Unless otherwise defined, otherwise all technology used herein and scientific terminology all have with those skilled in the art and usually understand identical implication.Although can use any method, device and the material similar or equivalent with person described herein in practice of the present invention or test, describe now preferred method, device and material.
Term " rigorous hybridization conditions " means low ionic strength known in affiliated field and the condition of high temperature.Usually under rigorous condition, but but the detection level of probe and its target sequence hybridization than (for example surpassing at least 2 times of backgrounds with the detection level of other sequence hybridization is higher.Rigorous hybridization conditions is sequence dependent, will be different under different envrionment conditionss, and sequence specific hybrid under comparatively high temps of growing.The preciseness of hybridizing by control or wash conditions can be identified the target sequence with probe 100% complementation.Detailed guidance for nucleic acid hybridization can be with reference to related documents (Tijssen, Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Probes, " Overview of principles of hybridization and the strategy of nucleic acid assays.1993).More specifically, described rigorous condition is selected as usually lower than the heat fusion joint (T of distinguished sequence under regulation ionic strength pH
m) approximately 5-10 ℃.T
mFor under equilibrium state 50% with the probe hybridization of target complementation during to target sequence residing temperature (under specify ion intensity, pH and nucleic acid concentration) (because of the excessive existence of target sequence, so at T
mUnder under equilibrium state 50% probe be occupied).Rigorous condition can be following condition: wherein in 7.0 to 8.3 times salt concn of pH lower than about 1.0M Na ion concentration, be generally approximately and 0.01 arrive 1.0M Na ion concentration (or other salt), and temperature is at least about 30 ℃ for short probe (including, but is not limited to 10 to 50 Nucleotide), and is at least about 60 ℃ for long probe (including, but is not limited to greater than 50 Nucleotide).Rigorous condition also can realize by adding such as the destabilizing agent of methane amide.For selectivity or specific hybrid, positive signal can be the background hybridization of twice at least, is 10 times of background hybridizations according to circumstances.Exemplary rigorous hybridization conditions can be as follows: 50% methane amide, and 5 * SSC and 1%SDS cultivate under 42 ℃; Or 5 * SSC, 1%SDS cultivates under 65 ℃, washs in 0.2 * SSC and washs in 0.1%SDS under 65 ℃.Described washing can be carried out 5,15,30,60,120 minutes or the longer time.
Term " recombinant host cell strain " or " host cell " mean to comprise the cell of polynucleotide of the present invention, and no matter use which kind of method to insert to produce recombinant host cell, for example directly absorb, other known method in transduction, f pairing or affiliated field.Exogenous polynucleotide can remain the nonconformity carrier of plasmid for example or can be integrated in host genome.Host cell can be prokaryotic cell prokaryocyte or eukaryotic cell, and host cell also can be unifacial leaf or dicotyledons cell.
Term " polynucleotide " means deoxyribonucleotide, dezyribonucleoside, ribonucleoside or ribonucleotide and the polymkeric substance thereof of sub-thread or bifilar form.Unless specific limited, otherwise the nucleic acid of the known analogue that contains natural nucleotide contained in described term, described analogue have the binding characteristic that is similar to reference nucleic acid and carry out metabolism in the mode of the Nucleotide that is similar to natural generation.Unless other specific limited, otherwise described term also means oligonucleotide analogs, it comprises PNA (peptide nucleic acid(PNA)), DNA analogue (thiophosphatephosphorothioate, phosphamide acid esters etc.) used in antisense technology.Unless otherwise, otherwise specific nucleic acid sequence is also impliedly contained its conservative varient of modifying (including, but is not limited to degenerate codon replaces) and complementary sequence and the clear and definite sequence of appointment.Specific, can realize that through mixing sequence that base and/or Hypoxanthine deoxyriboside residue replace degenerate codon replaces (people such as Batzer, Nucleic Acid Res.19:5081 (1991) by producing one of them or selected more than one (or all) codon the 3rd; The people such as Ohtsuka, J.Biol.Chem.260:2605-2608 (1985); With the people such as Cassol, (1992); The people such as Rossolini, Mol Cell.Probes 8:91-98 (1994)).
Term " polypeptide ", " peptide " and " albumen " in this article Alternate to mean the polymkeric substance of amino-acid residue.That is, be equally applicable to describe peptide and describe albumen for the description of polypeptide, and vice versa.Described term is applicable to natural generation aminoacid polymers and one of them or the aminoacid polymers that above amino-acid residue is non-naturally encoded amino acids.As used herein, the amino acid chain of any length contained in described term, and it comprises full-length proteins (being antigen), and wherein amino-acid residue connects via the covalency peptide bond.
Description of drawings
The total RNA of Fig. 1 autumn eggplant extracts figure.
Fig. 2 degenerate pcr amplification; M:Marker 1: the degenerate pcr result.
Figure 33 `RACE-PCR amplification; M:Marker 1:PCR result.
Figure 45 `RACE-PCR amplification; M:DL2000Marker 1:PCR result.
The amplification of Fig. 5 QqERF cDNA full length sequence; M:DNA Marker 1:PCR product.
The systematic evolution tree analytical results of Fig. 6 QqERF aminoacid sequence; A. Arabidopis thaliana; B. capsicum; C. Radix Dauci Sativae; D. upland cotton; E. soybean; F. small bell stings; G. tomato; H. paddy rice; I. the autumn eggplant; J. potato.
The structure schema of Fig. 7 QqERF Subcellular Localization carrier p163-QqERF-GFP.
The structure schema of Fig. 8 QqERF transcriptional activation effect plasmid pbridge-QqERF.
Fig. 9 QqERF protein subcellular positioning result; A:p163-QqERF-GFP; B: contrast: p163-GFP.
Figure 10 QqERF transcriptional activation function the result.
The structure schema of Figure 11 expression vector pC2301-35s-QqERF.
The structure schema of Figure 12 expression vector pC2301-rd29A-QqERF.
Figure 13 recombinant plasmid pC2301-35s-QqERF colony PCR amplification result; M:mark; 1-20: bacterium colony sample; CK: negative control; CK+: positive control.
Figure 14 recombinant plasmid pC2301-rd29A-QqERF colony PCR amplification result; M:mark; 1-20: bacterium colony sample; CK: negative control; CK+: positive control.
The enzyme of Figure 15 recombinant plasmid pC2301-35s-QqERF is cut result; 1.XbaI single endonuclease digestion; 2, XbaI+KpnI double digestion; M:mark; 1, plasmid; 2, XbaI single endonuclease digestion; 3, XbaI+KpnI double digestion.
The enzyme of Figure 16 recombinant plasmid pC2301-rd29a-QqERF is cut result; 1.XbaI single endonuclease digestion; 2, XbaI+KpnI double digestion; 3, PstI+XbaI double digestion.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiment are only exemplary, scope of the present invention are not consisted of any restriction.It will be understood by those skilled in the art that lower without departing from the spirit and scope of the present invention and can modify or replace details and the form of technical solution of the present invention, but these modifications and replacement all fall within the scope of protection of the present invention.
The checking of embodiment 1 autumn eggplant ERF transcription factor (QqERF) gene cloning, bioinformatic analysis and structural domain
1 materials and methods
1.1 material
1.1.1 vegetable material is cultivated and Stress treatment
Autumn eggplant branch is collected in Shenzhen Mangrove woods wilderness area., win young leaflet tablet and process with liquid nitrogen immediately Bruguiera conjugata spray immersion treatment 5h with the NaCl of 250mM, be stored in-80 ℃ of Ultralow Temperature Freezers standby.
1.1.2 bacterial strain and plasmid
1.1.3 enzyme and reagent
Plant total RNA extraction reagent box and DNA glue reclaim test kit and high pure plasmid prepares test kit in a small amount all available from Beijing hundred Tyke Bioisystech Co., Ltd; The Taq enzyme, 3 ' full RACE Kit and 5 ' full RACE Kit are available from TaKaRa company; Restriction enzyme, T4 ligase enzyme etc. are available from NEB company; The first chain cDNA synthetic agent box is available from MBI company.Primer is synthetic to be completed by Beijing AudioCodes Bioisystech Co., Ltd with gene sequencing.
Chloroform, Virahol, dehydrated alcohol, primary isoamyl alcohol, peptone, yeast extract, NaCl, MgCl
2The saturated phenol of Tris, SODIUM PHOSPHATE, MONOBASIC etc. are domestic analytical reagent, penbritin, agarose, diethylpyrocarbonate (DEPC), isopropyl-β-D-thiogalactoside(IPTG) (IPTG), 5-bromo-4-chloro-3-indoles-β-D-galactoside (X-gal) philosophy is available from Beijing lark gram biotech firm.
1.1.4 solution
1.TE(pH?8.0)：10mmol/L?Tris-HCl；1mmol/L?EDTA(pH?8.0)。
2.50 * TAE (1L): Tris 242.0g; Glacial acetic acid 57ml; 0.5mMEDTA 100ml (pH 8.0).
3.1M (pH 8.0,500ml): Tris 60.6g for Tris-HCl; Water 400ml; Dense HCl 21.0ml.
4.10 * TE damping fluid, 1 * TE/LiAc, salmon sperm dna (10mg/L), PEG/LiAc, Z-damping fluid, Z-Buffer/X-gal solution, ONPG (ortho-nitrophenyl β-D-galactoside).
5.0.1M spermidine, 60mg/ml tungsten powder suspension, dehydrated alcohol, 70% alcohol.
1.1.5 main experimental implementation instrument
Pcr amplification instrument (Bio-RAD),-80 ℃ of Ultralow Temperature Freezers (SANYO), high speed freezing centrifuge (Hettich), high speed tabletop centrifuge (Hettich), electrophoresis equipment (Bio-RAD), gel imaging system (Bio-RAD), Leica TCS SP2Confocal Spectral Microscope (UV-VIS) laser confocal microscope, the MK-20 dry-type thermostat, PDS-1000/He
TMThe type particle gun, water-bath, electro-heating standing-temperature cultivator, shaking table, high-pressure sterilizing pot etc.
1.1.6 substratum
1.1.6.1LB liquid nutrient medium (1L, pH 7.4): peptone 10.0g, yeast extract 5.0g, NaCl10.0g.
1.1.6.2LB solid medium (1L, pH 7.4): peptone 10.0g, yeast extract 5.0g, NaCl10.0g, agar powder 15.0g.
1.1.6.3MS substratum (1L, pH 5.8)
1.MS solid medium (1L):
2. stock solution I (1L)
3. stock solution II (1L)
CaCl
2·2H
2O 8800mg
4. stock solution III (1L)
FeSO
4·7H
2O 5560mg
Na
2-EDTA·2H
2O 7460mg
5. stock solution IV (1L)
6. stock solution V (1L)
Inositol 20000mg
1.2 experimental technique
1.2.1 the extraction of the total RNA of autumn eggplant (TRIzol method)
Extract the total RNA of autumn eggplant according to the TRIzol method;
1.2.2 synthetic (being undertaken by Invitrogen company reverse transcription test kit specification sheets) of the first chain cDNA
1. denaturation: take the total RNA of autumn eggplant as template, take Oligo-dT as primer (working concentration is 10pmol/ μ l).Reaction system such as table 1, after mixing, 70 ℃ of sex change 5min, be placed in 2min on ice immediately;
Table 1RNA denaturation system
2. reverse transcription: add mixing after following ingredients listed in the above-mentioned centrifuge tube, moment is centrifugal, 42 ℃ of 60min, and 70 ℃ of 10min deactivation ThermoScript II termination reactions ,-20 ℃ save backup;
Table 2 reverse transcription system
3.RNaseH digestion: add the RNaseH of 1 μ l (2U/ μ l) in the reaction solution, 37 ℃ of 20min, the single stranded RNA that digestion is combined with cDNA ,-20 ℃ of Refrigerator store reverse transcription products.
1.2.3AP2/ERF transcription factor family gene homologous fragment design of primers
1.2.3.1 the design of primer is with synthetic
Utilize DNAMAN software to carry out the homology analysis to Nucleotide and the aminoacid sequence of the relevant platymiscium AP2/ERF of the section genoid announced in Genbank, in conjunction with the synthetic pair of degenerate primers of territory conserved regions design, primer is by Beijing AudioCodes biotechnology limited liability company synthetic (seeing Table 3) according to this genoid DNA.
The primer that uses in table 3 degenerate pcr
1.2.3.2RT-PCR
Take the Kandelia candel mangrove leaf cDNA as template, use degenerated primer QEF and QER to carry out RT-PCR, the PCR condition is: 94 ℃ of 3min, (94 ℃ of 40sec, 56 ℃ of 40sec, 72 ℃ of 40sec) 30 circulations, 72 ℃ of 10min.Reaction is identified the PCR product with 2% agarose gel electrophoresis after finishing.
1.2.3.3PCR the recovery of product
Adopt the Easypure PCR Purification kit of Beijing Quanshijin Biotechnology Co., Ltd to reclaim the purpose fragment, operate according to the method for test kit specification sheets.
1.2.3.4 the purpose fragment is connected with cloning vector
The PCR product that reclaims is connected on the pMD19-T carrier, ligation system such as table 4,16 ℃ of connections are spent the night.
Table 4 ligation system
1.2.3.5 the competent preparation of intestinal bacteria
Carry out with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition, J. Pehanorm Brooker) book.
1.2.3.6 colibacillary conversion (heat shock method)
Carry out with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition, J. Pehanorm Brooker) book.
1.2.3.7 bacterium colony PCR identifies
White single bacterium colony with picking on the LB flat board that contains Amp changes 1mlLB (Amp over to
+) in liquid nutrient medium, 37 ℃, 200rpm shakes bacterium 2h, draws respectively 1 μ l bacterium liquid as template, carries out bacterium colony PCR and identifies, primer is SP1 and ASP2, amplification condition is: 94 ℃ of 5min; 94 ℃ of 30sec, 50 ℃ of 30sec, 72 ℃ of 30sec, 30 circulations; 72 ℃ of 10min.Reaction is identified the PCR product with 0.8% agarose gel electrophoresis after finishing.
1.2.3.8 the nucleotide sequencing of recombinant plasmid
Selecting picking white colony on substratum, carrying out bacterium colony and cultivate also PCR checking, primer is QEF and QER, and amplification condition is: 94 ℃ of 3min, (94 ℃ of 45sec, 55 ℃ of 45sec, 72 ℃, 40sec) 30 circulations, 72 ℃ of 10min.With 2% agarose gel electrophoresis, the PCR product is identified after reaction finishes, and screening is obtained positive colony send Beijing AudioCodes Bioisystech Co., Ltd to check order.
1.2.4.1 the design of primer is with synthetic
According to the homologous fragment of acquired ERF gene, the nido gene specific primer of two couples of 3 ' RACE of design, synthetic by Beijing AudioCodes biotechnology limited liability company, see Table 5.
Table 53 ' primer that uses in RACE reaction
1.2.4.2 the extraction of the total RNA of autumn eggplant
Adopt the TRIzol method to extract total RNA take Kandelia candel leaf as material.
1.2.4.3 the first chain cDNA's is synthetic
The same 1.2.2 of method.
1.2.4.4 nest-type PRC amplification 3 '-end cDNA
Take cDNA as template, use respectively gene specific primer QE3F1 and QE3F2 and 3 ' RACE reverse primer 3-outer and 3-inner to carry out 50 μ l system two-wheeled pcr amplifications, reaction parameter is: 94 ℃ of 3min, (94 ℃ of 45sec, 56 ℃ of 45sec, 72 ℃ of 60sec) 30cycles, 72 ℃ of 10min.50 times of first round PCR product dilutions are taken turns amplification template as second, at last amplified production is detected with 2% agarose gel electrophoresis, recovery PCR product also transforms bacillus coli DH 5 alpha, checks order after utilizing α complementation and bacterium colony PCR screening positive clone.
1.2.5 nest-type PRC amplification 5 ' end cDNA
1.2.5.1 the design of primer is with synthetic
According to two pairs of nido gene specific primers of acquired 3 ' end cDNA sequences Design, by the biological skill of Beijing AudioCodes limited liability company synthetic (table 6).
Table 65 ' primer that uses in RACE reaction
1.2.5.2 the first chain cDNA's is synthetic (according to the Invitrogen Gene Racer of company
TMThe explanation of kit test kit is carried out)
1. dephosphorization acid: add table 7 ingredients listed mixing in the PCR pipe, centrifugal, centrifugal after processing 1h under 50 ℃ of conditions, be placed in 1-2min on ice.
Table 7 dephosphorylation reaction system
2.RNA purifying
Carry out with reference to listed concrete grammar in " molecular cloning experiment guide " (third edition, J. Pehanorm Brooker) book.
3. remove cap: add table 8 ingredients listed mixing in the PCR pipe, moment is centrifugal, and after processing 1h under 37 ℃ of conditions, moment is centrifugal, is placed in 1-2min on ice.
Table 8 goes the cap reaction system
4.RNA purifying
5.5 ' RACE pre-treatment and reverse transcription
Use TaKaRa 5 '-Full RACE Kit to carry out CIAP, TAP to Kandelia candel leaf Total RNA 2ug and process, and with after 5 ' RACE Adaptor is connected, cDNA is synthesized in reverse transcription, sets up simultaneously M-MLV (-) to contrast (the same 1.2.2 of method).
1.2.5.3 nest-type PRC amplification 5 ' end cDNA
The cDNA that obtains take reverse transcription uses respectively gene specific primer QE5R1 and QE5R2 and 5 ' RACE forward primer 5-outer and 5-inner to carry out 50 μ l system two-wheeled pcr amplifications as template, and reaction system sees Table 9 and table 10.Two-wheeled PCR reaction conditions is: 94 ℃ of 3min, (94 ℃ of 45sec, 57 ℃ of 45sec, 72 ℃ of 2min) 30cycles, 72 ℃ of 10min.
Table 9Outer PCR reaction system
Table 10Inner PCR reaction system
3.0.8% agarose gel electrophoresis reclaims the PCR product and also is connected to the pMD19-T carrier, transforms bacillus coli DH 5, utilizes α complementation and bacterium colony PCR screening positive clone, and send the order-checking of AudioCodes order-checking section, the same 1.2.3.3-1.2.3.8 of method.
1.2.6 autumn eggplant ERF gene cDNA total length amplification
1.2.6.1 the design of primer is with synthetic
Splice known 3 '-end and 5 '-end cDNA sequence, obtain the cDNA full length sequence, accordingly, the design gene specific primer, the cDNA full length sequence of amplification autumn eggplant ERF gene, the primer sees Table 11.
1.2.6.2 the first chain cDNA's is synthetic
The same 1.2.2 of method.
The primer that the amplification of table 11cDNA full length sequence is used
1.2.6.3 clone and the bioinformatic analysis of autumn eggplant ERF gene cDNA full length sequence
1. obtain cDNA as template take the reverse transcription of QT primer, utilize gene specific primer QERFF and QERFR, carry out pcr amplification, reaction parameter: 94 ℃ of 5min; 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 2min, 30 circulations; 72 ℃ of 10min.
2.0.8% agarose gel electrophoresis reclaims the PCR product and is connected to the pMD19-T carrier, transforms bacillus coli DH 5, utilizes complementation and bacterium colony PCR screening positive clone, and order-checking obtains autumn eggplant ERF gene cDNA full length sequence, the same 1.2.3.3-1.2.3.8 of method.
3. utilize DNAMAN software to carry out sequence alignment and analysis; Utilize online ORF finder software analysis
Http:// www.ncbi.nlm.nih.gov/gorf/orfig.cgCarry out the open reading frame analysis; Utilize online SMART software
Http:// smart.emblheidelberg.de/smart/set_mode.cgi? GENOMIC=1Analyze typical structure territory, QqERF gene C DS district; Utilize online ExPASy Compute pI/Mw tool software
Http:// www.expasy.ch/tools/pi_tool.htmlQqERF albumen is carried out the prediction of physical properties; Utilize the three-D space structure of FeatureMap3D software analysis prediction QqERF albumen; Utilize NCBI-BLAST and DNAMAN software to carry out phylogenetic analysis.
1.2.7QqERF the checking of structural domain
1.2.7.1QqERF the Subcellular Localization of transcription factor
Transcription factor is all to work in nucleus usually.QqERF has the nuclear localization sequence of prediction.They may be positioned at the prompting of this constructional feature nucleus and work as consideration convey record regulon.This test builds green fluorescent protein (green fluorescent protein, GFP) fusion expression vector p163-QqERF-GFP, take empty carrier p163-GFP as contrast, Bombardment-Mediated Transformation onion epidermis cell, laser scanning co-focusing microscope are observed the Subcellular Localization situation of checking QqERF albumen.
1. design of primers is with synthetic
Synthetic primer GFP HQ (F) and the GFPHQ (R) that contains respectively restriction enzyme site Hind III and BamH I of design, sequence sees Table 12
Table 12 Subcellular Localization the primer
2.GFP the structure of fusion expression vector
Utilize primer GFPHQ (F) and GFPHQ (R) to amplify complete open reading frame from the pMD19-T carrier that is connected with total length QqERFcDNA.Amplified production and p163-GFP carrier are after double digestion, agarose gel electrophoresis, recovery, and the T4 ligase enzyme is connected in QqERF in Hind III and the BamHI site of p163-GFP carrier, builds the expression vector p163-QqERF-GFP that merges with the GFP translation.
3. the conversion of recombinant plasmid and bacterium colony PCR identify
The same 1.2.3.3-1.2.3.8 of method.
4. a small amount of of recombinant plasmid extraction (alkaline process) and enzyme are cut evaluation
Method prepares in a small amount test kit with reference to hundred Tyke plasmids and TaKaRa company enzyme is cut explanation, and the enzyme system of cutting sees Table 13.
Table 13 endonuclease reaction system
5. Bombardment-Mediated Transformation onion epidermis cell
5.1 material is prepared
Onion entocuticle (layer 5) is cut into 0.5cm * 0.5cm fritter, and the back of tearing faces up, and puts the substratum in MS, preculture 4h.
5.2 bronze suspension preparation
1) take 20mg (40 rifle) 1.0 μ mol/L bronzes, put into the 1.5ml centrifuge tube, add 500 μ l dehydrated alcohols, vortex vibration 1min, the centrifugal 1min of 10000r/min abandons supernatant, repeats 3 times;
2) add the distilled water of 500 μ l sterilizations, vortex vibration 1min, the centrifugal 1min of 10000r/min abandons supernatant, repeats 2 times;
3) add the distilled water of 400 μ l sterilizations, vortex vibration suspension bronze, at the vortex vibrator at a slow speed in non-stop-machine situation, in bronze evenly distribute to 10 a 0.5ml centrifuge tube, every pipe 40 μ l, 4 ℃ of preservations;
5.3 particle gun particulate bullet preparation
1) DNA, bronze (diameter is 1.0um), spermidine, CaCl
2According to the form below mixes:
Plasmid DNA bronze suspension 0.1mol/L 2.5mol/L
(5ug/ul) (50mg/ml) spermidine CaCl
2
2μl 6μl 4μl 6μl
2) mix vibration mixing 3min;
3) standing 15min on ice;
4) the centrifugal 10sec of 12000r/m (referring to that rotating speed reaches 12000 rear 10sec);
5) abandon supernatant, add the thick vibrations of 140 μ l dehydrated alcohols rear (breaing up bronze), the centrifugal 10sec of 12000r/m collects the bronze precipitation;
6) add 20 μ l dehydrated alcohols to suspend and precipitate, stand-by.
5.4 transform
1) open the power switch of vacuum pump and particle gun;
2) open the helium tank valve, rotation helium governor lever, making air pressure is 1300psi;
3) screw off with special spanner and can split the film door, can split film and be placed on door central authorities, door is screwed with spanner;
4) little bullet launching device is shifted out the bombardment chamber, screw off lid, put into and stop net, the particulate slide glass is arranged on (fine-grained one faces down) in pickup groove, screw on lid, the particulate launching device is put back to the bombardment chamber;
5) sample disc is placed on the appropriate location of bombardment chamber, aims at the target area, shut and bombard the chamber door;
6) pressing the VAC switch vacuumizes, when the vacuum meter numerical value when the lower left is (27-28in Hg), switch is got to HOLD switch place, then pin the FIRE switch, until get to suitable pressure, hear that can split the film sound that automatically breaks unclamps the FIRE switch again, simultaneously switch is got to the VENT place, to discharge the indoor vacuum of bombardment;
7) treat that vacuum gauge pressure is zero, and after the FIRE pilot lamp goes out, open bombardment chamber door, rotate culture dish;
8) take out little bullet launching device, unload particulate slide glass and barrier, the second shotgun that more renews, and the barrier that unloads and particulate slide glass pickup groove are put into 70% alcohol immersion;
9) screw off and can split the film door, elimination can be split the film fragment, again loads onto new split film, and screws on;
10) repeat 5-7,2 rifles/ware finishes, and opens and bombards the chamber door, takes out culture dish, the ware that closes lid, and sealing;
11) the same repetition of next ware material operation 3-10 is until bullet has been beaten;
12) turn off the helium tank main valve, pin the FIRE switch, bleed off the helium pressure in the gas acceleration tube, turn off power supply;
13) with the culture dish sealing, with black plastic bag parcel, be put in 25 degree incubators and secretly cultivate 24-36h.5.5Leica TCS SP2 confocal laser scanning microscope result
1.2.7.2QqERF the checking of transcription factor transcriptional activation function
Build pBridge-QqERF yeast effect plasmid, change in yeast strain AH109, and respectively at the single defective substratum of the SD that lacks tryptophane with lack on the SD substratum of the two defectives of SD of tryptophane, Histidine and screen positive transformant, carry out simultaneously that the colour developing of Whatman filter paper is identified and betagalactosidase activity detects tentatively and has transcriptional activation function by these two transcription factors of yeast one-hybrid verification experimental verification.
1. the design of primer is with synthetic
Design synthetic primer QEZ (F) and QEZ (R), sequence sees Table 14.
The primer that uses in table 14 yeast expression
2. the structure of effect plasmid
Amplify the gene complete open reading frame with primer QEZ (F) and QEZ (R) from the pMD19-T carrier that is connected with QqERF cDNA.Amplified production and pBridge are after double digestion, agarose gel electrophoresis, recovery, the T4 ligase enzyme is connected to target gene respectively EcoRI and the SalI site of pBridge carrier in the Matchmaker Yeast system, builds yeast fusion effect plasmid pBridge-QqERF.
3. transform and bacterium colony PCR evaluation
The same 1.2.3.3-1.2.3.8 of method.
4. a small amount of of recombinant plasmid extraction (alkaline process) and enzyme are cut evaluation
Carry out EcoRI and the reaction of SalI double digestion with the plasmid through detected through gel electrophoresis, the endonuclease reaction system is with table 15.
Table 13 endonuclease reaction system
5. the preparation of yeast competent cell and conversion
With reference to listed Lithium Acetate conversion method in " fine works molecular biology experiment guide " F. this uncle's one book difficult to understand.
6. betagalactosidase activity filter paper analysis and betagalactosidase activity are measured
With reference to listed method steps operation in " fine works molecular biology experiment guide " F. this uncle's one book difficult to understand.
2 experimental results and analysis
2.1ERF the acquisition of DNA homolog fragment
The ERF protein sequence of having reported has been carried out the homology compare of analysis, the DNA that finds ERF albumen presents the characteristic of high conservative in different plants, different ERF family in conjunction with the aminoacid sequence in territory, therefore utilize this conservative region to carry out the design of degenerated primer, the cDNA that the total RNA reverse transcription of blade of processing take salt stress obtains utilizes the degenerated primer of design to go out the approximately object tape of 200bp-300bp (Fig. 2) by pcr amplification as template.The PCR product that reclaims is connected on the pMD19-T carrier, transforms bacillus coli DH 5 alpha, utilize α complementation and bacterium colony PCR to filter out positive colony, obtain the middle conserved sequence of 270bp after order-checking.Show by BLAST and DNAMAN software analysis, the ERF gene nucleotide series of this sequence and Arabidopis thaliana, tobacco, wheat, paddy rice, soybean, corn has higher consistence, judges that tentatively this fragment is Halimodendron halodendron ERF gene conserved regions.
2.2ERF the acquisition of gene cDNA 3 ' sequence and 5 ' sequence
On acquired ERF DNA homolog fragment basis, design 3 ' RACE special primer, utilize gene specific primer QE3F1 and QE3F2 and 3 ' RACE reverse primer 3-Outer and 3-Inner, carry out the two-wheeled pcr amplification, electrophoresis detection obtains the approximately amplified production of 700bp (Fig. 3), sequencing result shows that this fragment length is 763bp, and 5 ' terminal sequence have respectively 35bp and former homologous fragment overlapping.
By homologous fragment and 3 ' the end fragment sequence that has obtained, design special primer QE5R1 and QE5R2, carry out 5 ' RACE amplification in conjunction with 5 ' RACE forward primer 5-outer and 5-inner again, electrophoresis detection obtains respectively the amplified production (Fig. 4) of two treaty 500bp and 200bp.
Reclaim respectively this two specific fragments, a plurality of positive transformed clones order-checkings of picking, result shows that the sequence that the 200bp sequencing fragment obtains is 5 ' the correct fragment of RACE, sequencing result is 176bp, and 3 ' terminal sequence have respectively 28bp and former homologous fragment overlapping.And experimental design M-MLV (-) contrast is further got rid of genomic dna and is polluted the false positive results that causes.
2.3 the acquisition of autumn eggplant ERF gene cDNA full length sequence
With 5 ' end and 3 ' end and homologous sequence splicing, design full length gene special primer QERFF and QERFR carry out pcr amplification, obtain 1200bp left and right product band (Fig. 5).Sequencing result shows, this full length sequence is 1209bp, and the sequence alignment result shows that the gene with various plants ERF family has higher similarity, is inferred as new ERF gene, called after QqERF.
2.4 autumn eggplant ERF cDNA full length sequence is analyzed
2.4.1ERF the sequence signature analysis of gene and the prediction of the structure function of proteins encoded
Using the cDNA full length sequence of ORF finder software analysis QqERF gene is 1209bp, complete open reading frame 1425bp (32bp-904bp), 290 amino acid of encoding, 5 '-UTR (1bp-31bp), 3 '-UTR (905bp-1209bp); SMART software is analyzed the protein structure domain of QqERF transcription factor coding, and result shows that QqERF albumen contains a typical AP2/EREBP structural domain that is positioned at the 54-117 position, is comprised of 64 amino acid, and two conservative elements of YRG and RAYD are arranged.YRG is conducive to the combination with DNA, the conformation that RAYD can be by affecting the YRG district or by regulating the combination of AP2 structural domain and DNA with the interaction of other albumen.The 14th and 19 amino acids of this conservative region are respectively conservative L-Ala (A) and aspartic acid (D); Be 8.09 by ExPASy Compute pI/Mw tool software prediction QqERF albumen iso-electric point, molecular weight is 31.5kDa.
2.4.2ERF the three-D space structure of albumen prediction
Utilize in the coded albumen space structure of the three-D space structure of FeatureMap3D software analysis prediction QqERF albumen: QqERF to have respectively 1 α spiral, 3 β-pleated sheet structures form, and this is the space structure that typical ERF class transcription factor has.
2.4.3QqERF the sequence analysis of albumen and other ERF2 proteinoid and systematic evolution tree analysis
Search AP2/ERF family clone gene mRNA sequence in NCBI, carry out gene order comparison and systematic evolution tree analysis with DNAMAN software, most crop ERF class transcription factor amino acid conserved regions such as result demonstration autumn eggplant and paddy rice, upland cotton, capsicum, tomato, soybean, Radix Dauci Sativae, Arabidopis thaliana, potato have higher homology.Sequence alignment result (seeing Fig. 6) shows: the crop clusters such as autumn eggplant ERF gene and Arabidopis thaliana, paddy rice together, and tomato, Radix Dauci Sativae, upland cotton, soybean, small bell thorn cluster are together, thereby in visible different plant species, still there is certain difference in same gene sequence molecular evolution, the inherent law that the evolution that can understand gene by comparison and the systematic evolution tree analysis of sequence homology and biosystem occur.
2.5QqERF the Subcellular Localization of transcription factor
2.5.1QqERF the structure (Fig. 7) of Subcellular Localization carrier p163-QqERF-GFP
2.5.2QqERF protein subcellular positioning result (Fig. 9)
2.6QqERF the checking of transcription factor transcriptional activation function
2.6.1QqERF the structure (Fig. 8) of transcriptional activation effect plasmid pbridge-QqERF
2.6.2QqERF transcriptional activation function the result
2.6.2.1QqERF the betagalactosidase activity filter paper colour developing result (Figure 10) of albumen in yeast
2.6.2.2QqERF the betagalactosidase activity analytical results (table 14) of albumen in yeast
The betagalactosidase activity detected result of table 14QqERF in yeast
The structure of 1 autumn of test example eggplant QqERF gene plant efficient expression vector and the checking of the correlation function in tobacco
1 materials and methods
1.1 material
1.1 vegetable material
Tobacco NC89 is by inventor's laboratory preservation
1.1.2 bacterial classification and carrier
Intestinal bacteria (Escherichia coli) DH5 α is by inventor's laboratory preservation;
Agrobacterium (Agrobacterium tumefaciens) bacterial strain LBA4404, the inventor preserves in the laboratory;
Dicotyledons expression vector pCAMBIA-2301 (illustrate: pCAMBIA2301 is laboratory plant expression vector commonly used, can find its complete sequence and explanation thereof on NCBI), the inventor preserves in the laboratory.
The pMD19-T cloning vector is available from TaKaRa.
1.1.3 reagent
Various restriction enzymes, rTaq enzyme, ExTaq enzyme are available from Takara company; T
4DNA ligase is available from Invitrogen company; The Taq enzyme is available from the Beijing Quanshijin Biotechnology Co., Ltd; Gel/PCR Extraction Kit is available from BioMIGA company.
1.1.4 substratum
1.YEB substratum:
With reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Pehanorm Brooker one book;
2.LB bacteria culture medium:
With reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Pehanorm Brooker one book;
3.MS substratum:
Referring to the listed concrete grammar of practical plant tissue culture technique study course (Gansu science tech publishing house) Cao Ziyi chief editor;
4.MS selection substratum:
Add 6-BA (3.0mg/L) in the MS minimum medium, NAA (0.2mg/L), Cephradine (500mg/L) and kantlex (100mg/L)
5.MS root media:
Add Cephradine (500mg/L) and kantlex (200mg/L) in the MS minimum medium
1.2 experimental technique
1.2.1 design of primers is with synthetic
Synthetic primer QqERF-XbaI (F) and QqERF-KpnI (R) and PstI-rd29A (F) and the rd29A-XbaI (R) that contains restriction enzyme site XbaI and KpnI of design.
Sequence sees Table 15.
Table 15 builds the plant expression vector the primer
1.2.2 the structure of plant expression vector
1.2.2.1 the structure of high efficiency plant expression vector pC2301-35S-QqRF
Utilize primer QqERF-XbaI (F) and QqERF-KpnI (R) to amplify complete open reading frame from the pMD19-T carrier that is connected with total length QqERFcDNA.Amplified production and carrier after double digestion, agarose gel electrophoresis, recovery, T
4Ligase enzyme is connected in QqERF in XbaI and the KpnI site of intermediate carrier pUCST4A, then downcut the expressed intact frame with HindIII and EcoRI double digestion, be connected to HindIII and the EcoRI site of pCAMBIA2301, build plant expression vector pC2301-35S-QqERF, transform bacillus coli DH 5 alpha, pcr amplification screening recombinant plasmid, and carry out corresponding enzyme and cut evaluation.
1.2.2.2 the structure of high efficiency plant expression vector pC2301-rd29A-QqERF
Utilize primer PstI-rd29A (F) and rd29A-XbaI (R) amplifying the promotor total length from the pMD19-T carrier that is connected with drought-inducible promoter rd29A, amplified production and carrier pCAMBIA2301 be T after PstI and XbaI enzyme cutting, agarose gel electrophoresis, recovery all
4Ligase enzyme connects, and connects product pC2301-rd29A again through XbaI and KpnI double digestion, with the QqERF purpose fragment T through XbaI and KpnI double digestion
4Ligase enzyme connects, and builds high efficiency plant expression vector pC2301-rd29A-QqERF, transforms bacillus coli DH 5 alpha, pcr amplification screening recombinant plasmid, and carry out corresponding enzyme and cut evaluation.
With reference to listed concrete grammar in " molecular cloning experiment guide " (third edition) J. Pehanorm Brooker one book;
1.2.2.3 a small amount of of recombinant plasmid is extracted and enzyme is cut evaluation
The plasmid extraction method prepares the listed concrete grammar of test kit in a small amount with reference to hundred Tyke plasmids; Enzyme is cut and is identified that TaKaRa company enzyme cuts explanation.
2 experimental results and analysis
2.1 the structure of plant expression vector
2.1.1 the structure schema of plant expression vector pC2301-35S-QqERF is seen Figure 11;
2.1.2 the structure schema of high efficiency plant expression vector pC2301-rd29A-QqERF is seen Figure 12.
2.1.3 the bacterium colony PCR of recombinant plasmid identifies
At the conversion LB of recombinant plasmid (Kan
+) on flat board, 20 single bacterium colonies of white of picking, carry out bacterium colony PCR and identify respectively, result is all positive, sees Figure 13 and 14.
2.1.4 the enzyme of recombinant plasmid is cut evaluation
The positive bacterium colony that bacterium colony PCR is detected changes LB (Kan over to
+) liquid nutrient medium, 37 ℃ are shaken bacterium and spend the night, and extract recombinant plasmid, carry out enzyme and cut evaluation, and recombinant plasmid pC2301-35S-QqERF and pC2301-rd29A-QqERF all cut out target stripe, and it is correct that enzyme is cut result, sees Figure 15 and 16.
After being transformed into ERF transcription factor cDNA of the present invention in tobacco, the Osmotic Adjustment Substances such as the proline(Pro) of transfer-gen plant have obvious rising, and the salt tolerance of transfer-gen plant is significantly improved.Experimental result shows, ERF transcription factor cDNA of the present invention can effectively improve the tolerance to environmental stress such as the Salt And Alkali Tolerance, disease and insect resistance of plant.
Claims (6)
1. the ERF transcription factor cDNA that separates from autumn eggplant [Kandelia candel (L.) Druce] is characterized in that, described cDNA is amino acid whose nucleotide sequence shown in coding SEQ ID No.2.
2. according to ERF transcription factor cDNA claimed in claim 1, it is characterized in that, described cDNA is the nucleotide sequence shown in SEQ ID No.1.
3. the coded albumen of the described ERF transcription factor cDNA of claim 1 or 2, is characterized in that, its amino acid is shown in SEQ ID No.2.
4. the expression vector that contains claim 1 or 2 described cDNA.
5. according to expression vector claimed in claim 4, it is characterized in that: described expression vector is plant expression vector.
6. the host cell that contains claim 4 or 5 described expression vectors.
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| CN103319584B (en) * | 2012-03-21 | 2015-01-21 | 深圳市农科集团有限公司 | Bruguiear gymnorrhiza (L.) Lam ERF transcription factor cDNA sequence, its expression vector and application |
| CN104293802B (en) * | 2013-09-23 | 2019-03-01 | 中国农业科学院生物技术研究所 | Crowtoe ERF class transcription factor, its encoding gene and expression vector and application |
| CN104313033B (en) * | 2013-09-23 | 2017-01-18 | 中国农业科学院生物技术研究所 | Lotis corniculatus L. stress resistance related transcription factor and coding gene and application thereof |
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| JP2003116546A (en) * | 2001-10-18 | 2003-04-22 | Denpatsu Kankyo Ryokka Center:Kk | Salt resistance-imparting protein, nucleic acid encoding the same, and salt-resistant transgenic plant |
| CN101565460A (en) * | 2008-04-21 | 2009-10-28 | 中国农业科学院生物技术研究所 | Populus euphratica DREB2 transcription factor cDNA sequence, expression carrier containing same and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2003116546A (en) * | 2001-10-18 | 2003-04-22 | Denpatsu Kankyo Ryokka Center:Kk | Salt resistance-imparting protein, nucleic acid encoding the same, and salt-resistant transgenic plant |
| CN101565460A (en) * | 2008-04-21 | 2009-10-28 | 中国农业科学院生物技术研究所 | Populus euphratica DREB2 transcription factor cDNA sequence, expression carrier containing same and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| 庞俊峰.红树科植物木榄和秋茄AP2/ERF和CaM基因的克隆研究.《中国优秀硕士学位论文全文数据库 农业科技辑》.2010,(第12期),12-36. * |
| 陈银华等.cDNA-AFLP法筛选红树植物盐应答基因.《中国农业科学》.2008,第41卷(第12期),4257-4263. * |
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