CN103319584A - Bruguiear gymnorrhiza (L.) Lam ERF transcription factor cDNA sequence, its expression vector and application - Google Patents

Abstract

Belonging to the field of plant genetic engineering, the invention discloses a Bruguiear gymnorrhiza (L.) Lam ERF (ethylene-responsive element binding factor) transcription factor cDNA sequence, its expression vector and application. The Bruguiear gymnorrhiza (L.) Lam ERF transcription factor cDNA sequence is the cDNA sequence that is separated and cloned from the mangrove plant Bruguiear gymnorrhiza (L.) Lam and encodes the ERF transcription factor, and the encoded amino acid sequence is shown as SEQ ID NO.3. The recombinant expression vector containing the ERF transcription factor nucleotide sequence involved in the invention is employed to transform tobaccos by an agrobacterium mediated conversion method, the plant leaf water content, the chlorophyll content, the conductivity, the malondialdehyde content and the like of a transgenic plant are determined, and the results show that the BgERF gene adjusts the content of osmotic adjustment substances so as to improve the salt tolerance of transgenic tobaccos and the normal physiological and biochemical functions of plants.

Description

Bruguiera conjugata ERF transcription factor cDNA sequence and expression vector thereof and application

Technical field

The present invention relates to a kind of cDNA sequence relevant with stress resistance of plant and the transcription factor of coding thereof, a cDNA sequence that relates in particular to from mangrove plant Bruguiera conjugata (Bruguiera gymnorhiza (L.) Lam) 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 plant genetic engineering field.

Background technology

Saline and alkaline is the main abiotic stress factor that affects terrestrial plant growth and restriction crop yield.In 1.3 hundred million hectares of cultivated areas of China, salt affected soil (saline soil) total area reaches 9,913 ten thousand hectares, and increases progressively in addition trend according to statistics.Salinization soil not only occupies very large proportion in China's cultivated area, other various countries also are like this in the world, for example according to the incomplete statistics of WHO (UNESCO) and the World Food Programme (FAO), interior 9.6 hundred million square kilometres of world wide, namely the land more than 30% is covered by saline-alkali soil, and is cumulative year after year trend; Half irrigation district, the whole world is subject to the impact of Secondary Saline and waterlogging to some extent.In the existing cultivated area of China, approximately there are 6,700,000 hectares to be subject to saline and alkaline harm at present, and along with process of industrialization is accelerated, the continuous expansion of irrigated land and plastic greenhouse area, the soil salinization area of China will further expand, this will seriously restrict China's agricultural to the utilization in soil, will have an immense impact on to agriculture production, ecological construction, Sustainable development simultaneously.

Salt damage has a strong impact on growth and development of plants, and ecotope is gone from bad to worse, and production has caused huge harm to agroforestry.Therefore, along with reduce and being on the rise of salt marsh day by day in arable land in the world wide, in the face of China's population constantly increases, arable land and the liquid manure resource reduces day by day, environmental aspect sharply worsens, agriculture production more and more is subject to stern challenge, how by the salt resistance ability that improves crop increase grain and cash crop output, economize on resources and improve environment, become the great demand of China's Sustainable Agricultural high-efficient development.And existing farm crop mostly belong to non-halophytes.How to make non-Halo crop can guarantee high yield again by normal growth in the salt marsh habitat, this is one of focus of for a long time scientists study.Face population increase in the world today, under the severe situation that limited and effective cultivated area constantly reduces, develop large-area saltings, cultivate the salt tolerant crop new variety and from then on go on the arena of history, become one of solution world food and energy starved effective way.

In the agriculture production core content of mankind's activity be constantly improve the environment of plant growth and constantly more new variety adapt to changeable ecotope, make the human how better product that obtains.Along with finishing successively of various plants genome blueprint examining order, carry out the research of the degeneration-resistant mechanism of coercing at plant at molecular level, understand the patience mechanism of plant; Excavate and the clone plant anti contravariance related gene, be conducive to genetic resources and carry out plant species improvement, create salt tolerant, the abiotic stress crop new germ plasms such as drought resisting for the resistance that improves farm crop, enlarge arable area, increase crop yield, will become the inexorable trend that the future of agriculture is produced.

Mangrove forest generally includes shrub and the arbor that is grown in the torrid zone, tideland, tidal swamp, subtropics and subtidal zone shoaling water, the living environment that has finally adapted to swampland tide bulge and fall in sea salt beach or the bay through very long Historical Evolution, periodically soak to flood and be rich in Weibull owing to being subject to seawater, become redness by the felling rear oxidation, therefore claim " mangrove ".The composition of mangrove forest is take kind rhizophoraceous as main, and Rhizophoraceae has 16 to belong to 120 kinds, and a part is grown in the inland, and a part forms mangrove forest, such as mangrove genus, Bruguiera conjugata genus, root of Kandelia genus, Common Ceriops genus etc.Be distributed in South East Asia, Africa and american torrid zone area.In China, tropical and subtropical zone seashore and the bay of the silting in the Hainan Island that mangrove forest mainly is distributed in, Guangxi, Guangdong and Fujian, or the alluviation solonchak at river outlet place or saliferous silty loam are suitable for the mangrove forest advolution.

Bruguiera conjugata [Bruguiera gymnorhiza (L.) Lam] belongs to the Rhizophoraceae Bruguiera conjugata and belongs to, and grows in the tideland, river mouth, bay that sea, Perenniporia martius land crosses." arbor or shrub, high 2-4m has stilit root, leaf keratin and alternately to life, ovalize.The flower both sexes, cyme, the fruit obovate is sagging, long 20-40cm.Bruguiera conjugata plays an important role in safeguarding coastal ecology balance, environmental monitoring, water body purification ".The living environment that they can adapt to sea wind erosion, salt solution proofing and anoxic substrate grows green and fresh all the year round.In the salt-tolerant plant molecular breeding, preferably go the theory of excavating to Saline Region based on the genetic material that improves the plant anti-salt performance.We clone the transcription factor gene that is closely related with salt tolerance from the mangrove Bruguiera conjugata, 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.

The plant stress-resistance genetically engineered is to import the discrete function gene to improve certain resistance, does not obtain improvement comprehensive, essence 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, in plant adverse circumstance signal transduction process, bringing into play crucial effect, 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.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) (comprising the 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 is comprised of 60～70 amino-acid residues, comprise 3 β-pleated sheet structures and 1 α spiral, by with goal gene upstream promoter zone in be rich in GC cis-acting elements (such 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.

Found at present to exist in plant materials a large amount of transcription factors, Arabidopis thaliana (A rabidopsis thaliana) only contains 27000 genes, is the encoding transcription factor with regard to 5.9% gene is arranged wherein.So far in Arabidopis thaliana, find 124 ERF family genes, in paddy rice, find 139 ERF family genes, also from other plant, isolate this class family gene such as tobacco, tomato, corn etc. in recent years.Only there is part to carry out functional analysis in the ERF gene of successfully cloning and reporting, yet in the Rhizophoraceae plant, there is not yet report.

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.There is report Tsil to do mutually in conjunction with territory and cis element GCC-box and DRE by its ERF, improved simultaneously salt tolerance and the disease resistance of transgene tobacco; Degeneration-resistant evidence tomato JERFs gene overexpression in tobacco can activate the expression of the degeneration-resistant functional gene of tobacco middle and lower reaches, improve transgene tobacco salt tolerance, drought tolerance, resistance to cold and disease resistance "; therefore; clone the new ERF class transcription factor gene with independent intellectual property right; study its basic biological characteristics and function; will provide fundamental basis for whole plant stress-resistance gene regulatory network and stress response reaction mechanism, for Crop Improvement resistance, the new degeneration-resistant material of creation provide material base.

In sum, for a water scarcity, salinization soil area constantly enlarge, cultivated area is limited, populous large agricultural country, how to utilize existing salinization to plough and limited middle-and-low-yielding fields, utilize genetic engineering means, be cloned in the regulatory gene that plays an important role in the plant stress-resistance process, obtaining the resistance and high-yielding new crop varieties is the available strategy that improves the comprehensive anti-adversity ability of farm crop, be related to national economy, the development of Sustainable development of national economy and agricultural is had far reaching significance.

Summary of the invention

One of purpose of the present invention is to provide a kind of Bruguiera conjugata ERF transcription factor.

A kind of Bruguiera conjugata ERF transcription factor of the present invention, called after BgERF is characterized in that the aminoacid sequence of described transcription factor contains the sequence shown in the SEQ ID NO.3.

What deserves to be explained is, with the replacement of the aminoacid sequence shown in the SEQ ID NO:3 by one or more amino-acid residues, disappearance or/and insert the aminoacid sequence that still has Bruguiera conjugata ERF functional transcription factor that obtains and also should drop within protection scope of the present invention.

Two of purpose of the present invention is to provide the cDNA sequence of the described transcription factor of coding.

The cDNA sequence of a kind of Bruguiera conjugata ERF transcription factor of encoding of the present invention, the cDNA sequence that it is from mangrove plant Bruguiera conjugata (Bruguiera gymnorhiza (L.) Lam) institute's coding ERF transcription factor of separating, cloning.

In specific embodiments of the invention, described cDNA sequence is shown in the SEQ ID NO:1 or shown in the SEQ ID NO.2.

What deserves to be explained is; with shown in the SEQ ID NO:1 or the one or more base of nucleotide sequence shown in the SEQ ID NO.2 replace, lack the nucleotide sequence that obtains or/and insert; if the coded albumen of this nucleotide sequence still has the function of Bruguiera conjugata ERF transcription factor, then this nucleotide sequence also should drop within protection scope of the present invention.

The present invention utilizes DNAMAN software that Nucleotide and the aminoacid sequence of the relevant platymiscium AP2/ERF of the section genoid announced among the Genbank are carried out the homology analysis, synthesize pair of degenerate primers according to this genoid DNA in conjunction with the territory conserved regions design, utilize the RT-PCR method from mangrove plant Bruguiera conjugata (Bruguiera gymnorhiza (L.) Lam), to be separated to the homologous fragment of AP2/ERF genoid, then obtain new AP2/ERF transcription factor gene BgERF (shown in the SEQ ID NO:1) by RACE-PCR technology clone, using the cDNA full length sequence of ORF finder software analysis BgERF gene is 1870bp, complete open reading frame 1428bp (262bp-1689bp, comprise initiator codon and terminator codon, shown in SEQ ID NO.2), 475 amino acid of encoding, 5 '-UTR (1bp-261bp), 3 '-UTR (1690bp-1870bp); SMART software is analyzed the protein structure domain of BgERF transcription factor coding, and the result shows that BgERF albumen contains a typical AP2/EREBP structural domain that is positioned at the 241-304 position, is comprised of 64 amino acid, and two conservative elements of YRG and RAYD are arranged.Utilize the three-D space structure of FeatureMap3D software analysis prediction BgERF albumen, the result shows: have respectively 1 α spiral in the coded albumen space structure of BgERF, 3 β-pleated sheet structures form, and this is the space structure that typical ERF class transcription factor has.

Three of purpose of the present invention is to provide a kind of plant expression vector of the cDNA sequence that contains described transcription factor and contains the Host Strains of this plant expression vector.

Described Host Strains can comprise bacillus coli DH 5 alpha and agrobacterium strains LBA4404.

In the specific embodiment of the invention, described plant expression vector is pBI121-BgERF.

Four of purpose of the present invention is to provide described Bruguiera conjugata ERF transcription factor and cDNA sequence thereof in the application that is improving in stress resistance of plant or the seed selection resistance crop, comprising: make up the plant expression vector that contains the above cDNA; Constructed plant expression vector is transformed in plant or the vegetable cell, cultivates the transgenic plant that obtain the tolerance to environmental stress raising.

Wherein, described resistance comprises that salt tolerant is coerced, drought-resistant or anti-low temperature.

To contain the plant expression vector of ERF transcription factor nucleotide sequence of the present invention by agriculture bacillus mediated conversion method transformation of tobacco, plant leaf water content to transfer-gen plant, chlorophyll content, specific conductivity and mda content etc. are measured, found that the plant leaf water content of transfer-gen plant, chlorophyll content all is higher than contrast, and the blade relative conductivity, mda and soluble sugar content all are lower than contrast, this five indices all becomes the important biochemical evidence that the transgenic plant saline-alkaline tolerance improves, thereby also further specify the BgERF gene improves transgene tobacco by the content of regulating osmotic adjustment salt tolerance, keep the normal biochemical functions of plant.

Description of drawings

Fig. 1 is the total RNA extraction of Bruguiera conjugata figure;

Fig. 2 is the degenerate pcr amplification;

M:Marker 1: the degenerate pcr result:

Fig. 3 is the 3`RACE-PCR amplification;

M:Marker 1:PCR result

Fig. 4 is the 5`RACE-PCR amplification;

M:DL2000Marker 1:PCR result

Fig. 5 is that BgERF cDNA full length sequence gets amplification;

M:DNA Marker 1:PCR product

Fig. 6 is the three-D space structure prediction of BgERF albumen;

Fig. 7 is the systematic evolution tree analytical results of BgERF aminoacid sequence;

A. Arabidopis thaliana; B. capsicum; C. the phase radish; D. upland cotton; E. soybean; F. small bell stings; G. tomato; H. Bruguiera conjugata; I. paddy rice; J. potato

Fig. 8 is the structure schema of BgERF Subcellular Localization carrier p163-BgERF-GFP;

Fig. 9 is the structure schema of BgERF transcriptional activation effect plasmid pBridge-BgERF;

Figure 10 is BgERF protein subcellular positioning result;

Figure 11 is BgERF transcriptional activation function the result;

Figure 12 is the structure schema of expression vector pBI121-BgERF;

Figure 13 is recombinant plasmid pBI121-BgERF colony PCR amplification result;

Figure 14 is that the enzyme of recombinant plasmid pBI121-BgERF is cut the result;

1.XbaI single endonuclease digestion; 2, XbaI+SacI double digestion

Figure 15 is the bacterium colony PCR that contains the Agrobacterium LBA4404 of expression vector pBI121-BgERF;

M:DL2000marker; +: positive control ,-: Agrobacterium LBA4404 (negative control)

Figure 16 is the PCR detected result that turns the dna level of BgERF genetic tobacco;

M:DL2000marker; +: positive control ,-: negative control

Figure 17 is that the PCR that turns the rna level of BgERF genetic tobacco detects

M:DL2000marker; +: positive control ,-: negative control

Figure 18 is the Salt Tolerance Analysis result of transgene tobacco;

Figure 19 is the impact of high salt pair plant water content;

Figure 20 is the impact of high salt pair mda content;

Figure 21 is the impact of high salt pair leaf soluble;

Figure 22 is the impact of high salt pair chlorophyll content in leaf blades;

Figure 23 is the impact of high salt pair blade specific conductivity.

Embodiment

Further describe by the following examples preparation method of the present invention and beneficial effect, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.

Illustrate: make the experimental methods of molecular biology specify in following examples, all with reference to " molecular cloning the experiment guide " (third edition, J. Pehanorm Brooker) listed concrete grammar carries out in the book, perhaps operates according to the reagent kit product specification sheets.

The checking of embodiment 1 Bruguiera conjugata ERF transcription factor (BgERF) gene cloning, bioinformatic analysis and structural domain

1 materials and methods

1.1 material

1.1.1 vegetable material is cultivated and Stress treatment

The Bruguiera conjugata 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 for subsequent use.

1.1.2 bacterial strain and plasmid

1.1.3 enzyme and reagent

Plant total RNA extraction reagent box Invitrogen Concert Plant RNA Reagent is available from U.S. hero's life technology company limited (Invitrogen); DNA glue reclaims 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 finished 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; 0.5mM EDTA 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 buffer, 1 * TE/LiAc, salmon sperm dna (10mg/L), PEG/LiAc, Z-Buffer,

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 SP2 Confocal Spectral Microscope (UV-VIS) laser confocal microscope, the MK-20 dry-type thermostat, PDS-1000/HeTM 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, NaCl 10.0g

1.1.6.2LB solid medium (1L, pH 7.4):

Peptone 10.0g, yeast extract 5.0g, NaCl 10.0g, agar powder 15.0g

1.1.6.3MS substratum (1L, pH 5.8)

1.MS solid medium (1L):

2. stock solution I (1L)

NH ₄NO ₃ 33000mg

KNO ₃ 38000mg

MgSO ₄·7H ₂O 7400mg

KH ₂PO ₄ 3400mg

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 Bruguiera conjugata

Operate according to Invitrogen Concert Plant RNA Reagent test kit, extract Bruguiera conjugata and organize the total RNA of blade, after the LiCl precipitation, use DNase I (RNase Free) (Code No.D2215) to carry out DNase I and process, finally be dissolved in RNA Free H ₂O is for subsequent use.

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 Bruguiera conjugata as template, take Oligo-dT as primer (working concentration is 10pmol/ μ l).Reaction system such as table 1,70 ℃ of sex change 5min place 2min on ice immediately behind the mixing;

Table 1RNA denaturation system

2. reverse transcription: add mixing after the 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 that Nucleotide and the aminoacid sequence of the relevant platymiscium AP2/ERF of the section genoid announced among the Genbank are carried out the homology analysis, 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 mangrove Bruguiera conjugata leaf cDNA as template, use degenerated primer MEF and MER to carry out RT-PCR, the PCR condition is: 94 ℃ of 3min, (94 ℃ of 45sec, 55 ℃ of 45sec, 72 ℃ of 45sec) 30cycles, 72 ℃ of 10min.Reaction is identified the PCR product with 1% 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

With white single bacterium colony of picking on the LB flat board that contains Amp, carry out bacterium colony and cultivate also PCR checking, primer is MEF and MER, amplification condition is: 94 ℃ of 3min, (94 ℃ of 45sec, 55 ℃ of 45sec, 72 ℃ of 40sec) 30 circulations, 72 ℃ of 10min.With 1% 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 the clone of 3 ' cDNA of Bruguiera conjugata ERF gene

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.

The primer that uses in table 53 ' RACE reaction

1.2.4.2 the extraction of the total RNA of Bruguiera conjugata

Adopt the TRIzol method to extract total RNA take the Bruguiera conjugata blade 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 ME3F1 and ME3F2 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.100 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 (as shown in table 6).

The primer that uses in table 65 ' RACE reaction

1.2.5.2 the first chain cDNA's is synthetic

Use TaKaRa 5 ' Full RACE Kit that Bruguiera conjugata blade Total RNA is carried out CIAP, TAP and process, and with after 5 ' RACEAdaptor is connected, cDNA is synthesized in reverse transcription, sets up simultaneously M-MLV (-) to contrast.(method is referring to TaKaRa 5 ' Full RACE Kit and the explanation of the Gene RacerTM kit of Invitrogen company test kit)

1.2.5.3 nest-type PRC amplification 5 ' end cDNA

The cDNA that obtains take reverse transcription uses respectively gene specific primer ME5R1 and ME5R2 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 7 and table 8.Two-wheeled PCR reaction conditions is: 94 ℃ of 3min, (94 ℃ of 45sec, 57 ℃ of 45sec, 72 ℃ of 2min) 30cycles, 72 ℃ of 10min.0.8% agarose gel electrophoresis reclaims the PCR product and is connected to the pMD19-T carrier, transforms bacillus coli DH 5 alpha, utilizes α complementation and bacterium colony PCR screening positive clone, and send the order-checking of AudioCodes order-checking section, the same 1.2.3.3-1.2.3.7 of method.

Table 7 Outer PCR reaction system

Table 8 Inner PCR reaction system

1.2.6 Bruguiera conjugata 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 the Bruguiera conjugata ERF gene that increases, the primer sees Table 9.

1.2.6.2 the first chain cDNA's is synthetic

The same 1.2.2 of method.

The table 9cDNA full length sequence employed primer that increases

1.2.6.3 clone and the bioinformatic analysis of Bruguiera conjugata ERF gene cDNA full length sequence

1. obtain cDNA as template take reverse transcription, utilize gene specific primer MERFF and MERFR, 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, reclaim the PCR product and be connected to the pMD19-T carrier, transform bacillus coli DH 5 alpha, utilize α complementation and bacterium colony PCR screening positive clone, and order-checking obtains Bruguiera conjugata 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.cg to carry out the open reading frame analysis; Do you utilize online SMART software http://smart.emblheidelberg.de/smart/set_mode.cgi? GENOMIC=1 analyzes typical structure territory, BgERF gene C DS district; Utilize online ExPASy Compute pI/Mw tool software http://www.expasy.ch/tools/pi_tool.html BgERF albumen to be carried out the prediction of physical properties; Utilize the three-D space structure of FeatureMap3D software analysis prediction BgERF albumen; Utilize NCBI-BLAST and DNAMAN software to carry out phylogenetic analysis.

1.2.7BgERF the checking of structural domain

1.2.7.1BgERF the Subcellular Localization of transcription factor

Transcription factor all is to work in nucleus usually.BgERF 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 makes up green fluorescent protein (green fluorescent protein, GFP) fusion expression vector p163-BgERF-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 BgERF albumen.

1. design of primers is with synthetic

Synthetic primer GFPHM-SalI (F) and the GFPHM-BamHI (R) that contains respectively restriction enzyme site SalI and BamHI of design, sequence sees Table 10.

Table 10 Subcellular Localization the primer

2.GFP the structure of fusion expression vector

Utilize primer GFPHM-SalI (F) and GFPHM-BamHI (R) to amplify complete open reading frame (shown in the SEQ ID NO.2 from the pMD19-T carrier that is connected with total length BgERF cDNA.)。Amplified production and p163-GFP carrier are after double digestion, agarose gel electrophoresis, recovery, and the T4 ligase enzyme is connected in BgERF in SalI and the BamHI site of p163-GFP carrier, make up the expression vector p163-BgERF-GFP that merges with the GFP translation.

3. the conversion of recombinant plasmid and bacterium colony PCR identify

The same 1.2.3.3-1.2.3.7 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 11.

Table 11 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 by weighing 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 that 500 μ l sterilize, vortex vibration 1min, the centrifugal 1min of 10000r/min abandons supernatant, repeats 2 times;

3) add the distilled water of 400 μ l sterilization, vortex vibration suspension bronze, at the vortex vibrator at a slow speed in the 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 (5ug/ul)	Bronze suspension (50mg/ml)	0.1mol/L spermidine	2.5mol/L?CaCl 2
				2μl	6μl	4μl	6μl

2) mix vibration mixing 3min;

3) leave standstill 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 suspension precipitation, stand-by.

5.4 method for transformation is with reference to Biolistic particle delivery system (the Model PDS-1000/He of BIO-RAD company) operation instructions.

5.5Leica TCS SP2 confocal laser scanning microscope result

1.2.7.2BgERF the checking of transcription factor transcriptional activation function

Make up pBridge-BgERF yeast effect plasmid, change among the yeast strain AH109, and screen positive transformant at the single defective substratum of the SD that lacks tryptophane and the SD substratum that lacks the two defectives of SD of tryptophane, Histidine respectively, 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 MEZ (F) and MEZ (R), sequence sees Table 12.

The primer that uses in table 12 yeast expression

2. the structure of effect plasmid

Amplify the gene complete open reading frame (shown in the SEQ ID NO.2 with primer MEZ (F) and MEZ (R) from the pMD 19-T carrier that is connected with BgERF cDNA.)。Amplified production and pBridge after double digestion, agarose gel electrophoresis, recovery, T ₄Ligase enzyme is connected to target gene respectively BamHI and the SalI site of pBridge carrier in the Matchmaker Yeast system, makes up yeast fusion effect plasmid pBridge-BgERF.

3. transform and bacterium colony PCR evaluation

The same 1.2.3.3-1.2.3.7 of method.

4. a small amount of of recombinant plasmid extraction (alkaline process) and enzyme are cut and are identified that the plasmid of using through detected through gel electrophoresis carries out BamHI and the reaction of SalI double digestion, and the endonuclease reaction system is with table 13.

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 RNA electrophoresis result of extraction as shown in Figure 1 with the total RNA of blade that salt stress is processed.) cDNA that obtains of reverse transcription is template, utilizes the degenerated primer of design to go out the approximately object tape of 200bp-300bp (shown in Figure 2) by pcr amplification.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 262bp after the 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.

2.2 the acquisition of Bruguiera conjugata ERF gene cDNA 3 ' sequence and 5 ' sequence

On acquired Bruguiera conjugata ERF dna homolog fragment basis, design 3 ' RACE special primer, utilize gene specific primer ME3F1 and ME3F2 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 (shown in Figure 3), sequencing result shows that this fragment length is 639bp, and 5 ' terminal sequence has respectively 86bp and former homologous fragment overlapping.

By homologous fragment and 3 ' the end fragment sequence that has obtained, design special primer ME5R1 and ME5R2, 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 (shown in Figure 4) of two treaty 1000bp and 500bp.

Reclaim respectively this two specific fragments, a plurality of positive transformed clones order-checkings of picking, the result shows that the sequence that the 1000bp sequencing fragment obtains is the correct fragment of 5 ' RACE, sequencing result is 969bp, and 3 ' terminal sequence has respectively 91bp and former homologous fragment overlapping.And experimental design M-MLV (-) contrast.

2.3 the acquisition of Bruguiera conjugata ERF gene cDNA full length sequence

With 5 ' end and 3 ' end and homologous sequence splicing, design full length gene special primer MERFF and MERFR carry out pcr amplification, obtain 1000-2000bp left and right sides product band (shown in Figure 5).Sequencing result shows, this full length sequence is 1870bp, 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 BgERF, and its nucleotide sequence is shown in SEQ ID NO.1.

2.4BgERFcDNA full length sequence analysis

2.4.1 the sequence signature analysis of Bruguiera conjugata ERF gene and the prediction of the structure function of proteins encoded

Using the cDNA full length sequence of ORF finder software analysis BgERF gene is 1870bp, complete open reading frame 1428bp (262bp-1689bp), 475 amino acid of encoding, 5 '-UTR (1bp-261bp), 3 '-UTR (1690bp-1870bp); SMART software is analyzed the protein structure domain of BgERF transcription factor coding, and the result shows that BgERF albumen contains a typical AP2/EREBP structural domain that is positioned at the 241-304 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 8.77 by ExPASy Compute pI/Mw tool software prediction BgERF albumen iso-electric point, molecular weight is 50.6kDa.

2.4.2BgERF the three-D space structure of albumen prediction

Utilize the three-D space structure (shown in Figure 6) of FeatureMap3D software analysis prediction BgERF albumen, as scheme to have respectively 1 α spiral in the coded albumen space structure of demonstration: BgERF, 3 β-pleated sheet structures form, and this is the space structure that typical ERF class transcription factor has.

2.4.3BgERF the sequence analysis of albumen and other ERF proteinoid and systematic evolution tree analysis

In NCBI, search for clone gene mRNA sequence of AP2/ERF family, carry out gene order comparison and systematic evolution tree analysis with DNAMAN software, the result shows that most crop ERF class transcription factor amino acid conserved regions such as Bruguiera conjugata and paddy rice, upland cotton, capsicum, tomato, soybean, Radix Dauci Sativae, Arabidopis thaliana, potato have higher homology.Sequence alignment result (as shown in Figure 7) shows: the crop clusters such as BgERF gene and Arabidopis thaliana, paddy rice together, and tomato, Radix Dauci Sativae, upland cotton, soybean, small bell thorn cluster are together, thereby still there is certain difference in same gene sequence molecular evolution in the visible different plant species, the inherent law that the evolution that can understand gene by comparison and the systematic evolution tree analysis of sequence homology and biosystem occur.

2.5BgERF the Subcellular Localization of transcription factor

2.5.1BgERF the structure of Subcellular Localization carrier p163-BgERF-GFP

The vector construction schema as shown in Figure 8.

2.5.2BgERF protein subcellular positioning result: the result as shown in figure 10.

2.6BgERF the checking of transcription factor transcriptional activation function

2.6.1BgERF the structure of transcriptional activation effect plasmid pbridge-BgERF

The vector construction schema as shown in Figure 9.

2.6.2BgERF transcriptional activation function the result

2.6.2.1BgERF the betagalactosidase activity filter paper colour developing result of albumen in yeast: the result as shown in figure 11.

2.6.2.2BgERF the betagalactosidase activity analytical results of albumen in yeast: the result is as shown in table 14.

The betagalactosidase activity detected result of table 14 BgERF in yeast

The structure of test example 1 Bruguiera conjugata BgERF gene plant efficient expression vector and the checking of the correlation function in tobacco

1 materials and methods

1.1 material

1.1.1 vegetable material

Tobacco NC89 is preserved by this laboratory

1.1.2 bacterial classification and carrier

Intestinal bacteria (Escherichia coli) DH5 α is preserved by this laboratory; Agrobacterium (Agrobacterium tumefaciens) bacterial strain LBA4404, preserve in this laboratory; Dicotyledons expression vector pBI121 (illustrate: pBI121 is laboratory plant expression vector commonly used, can find its complete sequence and explanation thereof on NCBI), preserve in this 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 substratum:

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 pBI121-XbaI (F) and the pBI121-SacI (R) that contains restriction enzyme site XbaI and SacI of design.Sequence sees Table 15.

Table 15 makes up 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 pBI121-BgERF

Utilize primer BgERF-XbaI (F) and BgERF-SacI (R) to amplify complete open reading frame (shown in the SEQ ID NO.2 from the pMD19-T carrier that is connected with total length BgERF cDNA.)。Amplified production and carrier after double digestion, agarose gel electrophoresis, recovery, T ₄Ligase enzyme is connected in BgERF in XbaI and the SacI site of carrier pBI121, replace the GusA gene on the carrier pBI121, make up plant expression vector pBI121-BgERF, transform bacillus coli DH 5 alpha, pcr amplification screening recombinant plasmid, and carry out corresponding enzyme and cut evaluation;

1.2.2.2 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 evaluation TaKaRa company enzyme and is cut explanation.

1.2.3 the genetic transformation of tobacco

1.2.3.1 the preparation of Agrobacterium LBA4404 competent cell and conversion (TSS method) method is referring to the preparation of fine works molecular biology experiment guide (Ao Sibai) single stage method and transformed competence colibacillus cell.

1.2.3.2 containing the PCR of the Agrobacterium of recombinant plasmid identifies

28 ℃ of YEB that cultivate 1-2d of picking (contain 50mg/L Kan respectively, 50mg/L Rif) the single bacterium colony on the conversion flat board, make negative control with unconverted Agrobacterium, with the positive contrast of plant expression carrier plasmid pBI121-35S-BgERF, carry out bacterium colony PCR and identify.

1.2.4 turn the detection of BgERF genetic tobacco

1.2.4.1DNA the PCR of level detects

The tobacco gene group DNA that utilizes the CTAB method to extract in a small amount to contain the Kan resistance detects the further positive tobacco plant of screening by PCR.If amplified target fragment, and do not amplify fragment in the negative control, namely the preliminary proof goal gene is incorporated in the tobacco gene group.

1.2.4.2RNA the PCR of level detects

To the tobacco plant of dna level test positive, utilize TRIzol reagent to extract total RNA, reverse transcription obtains cDNA as template, and RT-PCR detects the positive transformation seedlings of screening.Method is with reference to the 1.2.1-1.2.2 among the embodiment 1, PCR reaction parameter: 94 ℃ of 5min; 94 ℃ of 30sec, 60 ℃ of 30sec, 72 ℃ of 2min, 30 circulations; 72 ℃ of 10min; 0.8% agarose gel electrophoresis.

1.2.5 transgene tobacco anti-coerced analysis

1.2.5.1 the abiotic stress resistance of transgene tobacco is identified

Carry out the high-salt stress Resistance Identification with the tobacco plant of transplanting about 2 weeks of growth, establish three repetitions:

Salt tolerant is identified: watering in cultivation with the NaCl solution of 0mM, 120mM, 240mM respectively has in the basin of wild-type and transgenosis type tobacco 2 weeks of processing in 22 ℃ of illumination boxs; In 0h, 24h, 48h, 72h, 14d sampling, after the liquid nitrogen processing, place-80 ℃ of preservations respectively;

1.2.6 the Determination of Physiological And Biochemical Indices of transgene tobacco under the high-salt stress condition

1.2.6.1 the mensuration of plant leaf tissue water content behind the Stress treatment

To transgenosis and the non-transgenic tobacco of different concns Nacl Stress treatment, get blade at 0h, 24h, 48h, 72h, 14d respectively, clean with distilled water flushing, thieving paper blots surface-moisture.3 individual plants are got in each processing, and survey rapidly its fresh weight, put into afterwards 120 ℃ of baking ovens and toast 20min, and 80 ℃ of bakings are spent the night to constant weight, its dry weight of weighing.To above-mentioned dried, the fresh weight that records, adopt formula:

Tissue water content (accounting for fresh weight %)=(fresh weight-dry weight)/fresh weight carries out the calculating of water content.

1.2.6.2 the mensuration of mda (MDA) and soluble sugar

Take by weighing the tobacco leaf 0.5g through Stress treatment, liquid nitrogen freezing is also with after historrhexis's instrument fragmentation, add 1ml0.5%TBA, after the vortex concussion homogenate, again above-mentioned homogenate is transferred to clean 10mlEp pipe and numbering (contrast adds 1ml distilled water), and then add 0.5%TBA solution 5ml, in water-bath, boil behind the mixing and boil reaction 15min to sufficient reacting; Put into rapidly ice and cool off 10min, draw the supernatant low-speed centrifugal; Then survey respectively the absorbance of 532nm, 600nm and 450nm wavelength place supernatant liquor with spectrophotometer.

The calculation formula of mda (MDA) and soluble sugar content:

C (sugar)=11.71 * A450 (mmol/L)

C(MDA)＝6.45×(A532-A600)-0.56×A450(μmol/L)

Annotate: A450 in the formula, A532, A600 are respectively the liquid that records at 450nm, 532nm, the absorbance under the 600nm wavelength.

1.2.6.3 the transfer-gen plant chlorophyll content is analyzed

Take by weighing the tobacco leaf 0.2g through Stress treatment, after liquid nitrogen freezing is used historrhexis's instrument fragmentation, the acetone soln that adds 1ml 80%, vortex concussion homogenate is transferred to above-mentioned homogenate clean 10mlEp pipe and numbering (contrast adds 1ml distilled water) again, and then the acetone soln of adding 5ml80%, reaction 30min is until fragment of tissue is bleached low-speed centrifugal (4000r/min, 10min), get supernatant and survey A663, A645.

The calculation formula of chlorophyll content:

Ca＝12.7*A663-2.69*A645

Cb＝22.9*A645-4.68*A663

C _T＝Ca+Cb＝8.02*A663+20.21*A645

(annotate: C in the formula _T, Ca, Cb unit be mg/L)

1.2.6.4 rotaring gene plant blade is organized the relative conductivity analysis

Take by weighing tobacco leaf 0.2g through Stress treatment in the 10mlEP pipe, add the 5ml deionized water, natural immersion 1h measures specific conductivity J1 and record afterwards, then puts into boiling water bath and boils 10min, is cooled to room temperature and surveys specific conductivity J2 and record.

The calculation formula of plant tissue relative conductivity:

The * 100% of relative conductivity=(J1-J0)/(J2-J0)

(annotate: J0 represents the conductivity value of deionized water)

2 experimental results and analysis

2.1 the structure of plant expression vector

2.1.1 the structure schema of plant expression vector pBI121-BgERF is seen Figure 12;

2.1.2 the bacterium colony PCR of recombinant plasmid identifies

On the conversion LB of recombinant plasmid (Kan+) flat board, 20 single bacterium colonies of white of picking carry out bacterium colony PCR and identify respectively, and the result is all positive, sees Figure 13.

2.1.3 the enzyme of recombinant plasmid is cut evaluation

The positive bacterium colony that bacterium colony PCR is detected changes LB (Kan+) liquid nutrient medium over to, and 37 ℃ are shaken bacterium and spend the night, and extract recombinant plasmid, carry out enzyme and cut evaluation, and recombinant plasmid pBI121-BgERF cuts out target stripe, and it is correct that enzyme is cut the result, sees Figure 14.

2.2 containing the Agrobacterium PCR of expression vector detects

Recombinant plasmid pBI121-BgERF is transformed Agrobacterium LBA4404, make template with the bacterium liquid of the Agrobacterium after transforming, unconverted Agrobacterium makes negative control, and plasmid is done positive control, carries out bacterium colony PCR and identifies.Detected result shows, this recombinant plasmid successfully changes in the Agrobacterium, sees Figure 15.

2.3 turn the detection of BgERF genetic tobacco

2.3.1DNA the PCR of level detects

Choose Kan resistance seedling, its blade of clip adopts the CTAB method to extract its DNA, and take tobacco gene group DNA as template, take pBI121-XbaI (F) and pBI121-SacI (R) as primer, the employing round pcr carries out the detection of dna level to Kan resistance seedling.The result shows, in kan resistance seedling 22 strains of detection 16 strains positive, positive rate approximately 73%, plant PCR detects and sees Figure 16, proves that BgERF has been incorporated in the tobacco gene group.

2.3.2RNA the PCR of level detects

Choosing respectively the positive tobacco plant blade of 16 strains that dna level identifies is material, extracts total RNA, and the cDNA that obtains take reverse transcription is as template, and RT-PCR does further to detect to positive seedling.The result shows, 16 strains in the plant leaf of genomic dna-pcr test positive, 12 strain test positive, positive rate 75%, plant RT-PCR detect sees Figure 17.

2.4 transgene tobacco anti-coerced analysis

Salt Tolerance Analysis result: transfer-gen plant and the wild-type plant NaCl solution-treated of 240mM, wild-type plant 2 strain yellow leafs after 50 days, the stem stalk withers here to lodge and is tending towards dead, and surplus 1 strain survival is bloomed, and all survives and turn the BgERF gene plant, although plant is downgraded, blade is rare, but can normally bloom, and sees Figure 18, therefore it seems from morphological analysis, turn the salt tolerance that the BgERF gene plant has improved tobacco.

2.5 the Determination of Physiological And Biochemical Indices of transgene tobacco under condition of salt stress

2.5.1 salt stress is processed the impact on the plant leaf water content

The plant tissue water content is a common counter of expression plant tissue water regime.For the tissue of normal growth, the upgrowth situation that how much directly affects plant of water content.There are some researches show, high salt adverse circumstance all can cause the osmotic stress of plant materials, causes the cell dehydration, and serious caused cell turgor forfeiture causes death.

Table 16 240mM NaCl coerces lower plant leaf water content difference

This example is by measuring the variation of wild-type and transgenosis type plant water content under the stress conditions, thus the direct evidence that is significantly increased than wild-type plant salt tolerance at individual level acquisition transfer-gen plant.Result's demonstration, the water content of wild-type and transfer-gen plant was all on a declining curve after high salt (table 16, Figure 19) was processed.Before Stress treatment, the water content of the water content of transfer-gen plant and wild-type plant does not have notable difference; During 240mM NaCl Stress treatment 0-72h, the leaf water content of transgenosis type plant is starkly lower than the leaf water content of wild-type plant, when coercing 72h-14 days, the leaf water content of wild-type plant is reduced to 68.99%, obviously be subject to the impact of salt stress, and the leaf water content kept in balance of transgenosis type plant, still maintain about 89%, this shows the foreign gene BgERF of insertion, cell dehydration under the high-salt stress there has been certain mitigation, thereby has improved the salt tolerance of transgene tobacco under stress conditions.

2.5.2 salt stress is processed the impact on plant MDA content

Plant organ is old and feeble or when suffering the environment stress injury, tends to occur peroxidation of membrane lipids, and mda (MDA) is the final degradation production of peroxidation of membrane lipids, and its content can reflect that plant suffers the degree of adverse circumstance injury; Its generation can also aggravate the damage of film.Therefore, mda produces the degree that how much can represent the film lipid peroxidation of quantity, also can indirectly reflect the power of the resistance of oxidation of plant tissue.So in physiological responses of plants to anti-environment research, mda content is a common counter.

Table 17 240mM NaCl coerces the difference (unit: μ mol/g) of lower plant leaf MDA content

Can find out from table 17, Figure 20, wild-type type plant is after 240mM NaCl 48h salt stress is processed, just can significantly improve the MDA content of wild-type plant, and transfer-gen plant MDA content during 240mM NaCl coerces 0-14d increases slowly, when coercing 14 days, wild-type plant MDA content is about 1.5 times of transfer-gen plant.The MDA content analysis result of wild-type and transgenosis type plant shows, guarantees cell lactones membrane integrity thereby the BgERF transcription factor can reduce the adipose membrane peroxidation under environment stress.This may be owing to BgERF downstream signal and peroxylradicals resistance molecule (such as gsh, catalase, peroxidase etc.) phase coupling, thereby has protected cytolemma.

The variation of plant soluble sugar content after 2.5.3 salt stress is processed

In the environment of coercing, plant is in order to slow down the physiological metabolism imbalance that causes by coercing, a large amount of some small molecules organic compound of accumulation in the cell.Soluble sugar is one of small molecules solute of stress-inducing, reduces the flow of water by osmoregulation, keeps higher osmotic pressure, guarantees the normal physiological function of cell.

Table 18 240mM NaCl coerces the difference unit of lower plant leaf soluble sugar content: (mmol/g)

Can find out from table 18, Figure 21, when 240mM NaCl salt side of body 0-72h, soluble sugar content reduces in the plant, may be because under the salt stress of high density, its photosynthesis is subject to very large inhibition, and glucose is synthetic to be reduced, and causes soluble sugar content to reduce.Plant is being coerced between the 72h-14d, and the soluble sugar content in the body is with regard to bottom out, but not the increase of transfer-gen plant transfer-gen plant soluble sugar content will be higher than the increase (p＜0.05) of wild-type plant soluble sugar content.The soluble sugar content analytical results of plant shows, under the environment stress condition, thereby plant can guarantee normal metabolic process in the cell paste by spontaneous generation soluble sugar.Insert the transfer-gen plant of BgERF transcription factor, can under adverse environmental factor, produce acknowledgement mechanism, do not need to produce too many soluble sugar and resist environment stress.

2.5.4 the plant leaf chlorophyll content changed after salt stress was processed

Chlorophyll is that plant carries out photosynthetic basic substance, and chlorophyll content and leaf photosynthesis are closely related, and chlorophyll is comprised of chlorophyll a and chlorophyll b etc., and chlorophyll content and Net Photosynthetic Rate are proportionate.Under high salt adverse circumstance, because ion content is high in the plant leaf, make this combination become lax, its result makes more chlorophyll be dissociated out.Chlorophyll only has with chloroplast protein is combined, and just has the photochemically reactive function of absorption, conversion and generation to luminous energy.Under saline and alkaline adverse circumstance, dissociating of chlorophyll and chloroplast protein will make the active decline of chlorphyllase, thereby impel chlorophyll to decompose, and chlorophyll content in leaf blades is descended.

Table 19 240mM NaCl coerces the difference unit of lower plant leaf chlorophyll content: (mg/g)

Can find out from table 19, Figure 22, when 240mM NaCl salt side of body 0-72h, be in reduction trend before the chlorophyll content of wild-type and transfer-gen plant is coerced; And after coercing 14d, the chlorophyll content of transfer-gen plant returns to the front level of coercing; And the chlorophyll content of wild-type plant is still held downtrending.But the chlorophyll content analytical results of plant shows, under 240mM NaCl condition of salt stress, the photosynthesis of wild-type plant is subject to the impact of high salt, photosynthesis is suppressed, and the transfer-gen plant of insertion BgERF transcription factor, can produce acknowledgement mechanism at the environment stress initial stage, thereby guarantee the normal photosynthesis of plant strain growth.

The variation of plant leaf relative conductivity after 2.5.5 salt stress is processed.

Plant cell membrane plays an important role to microenvironment and the normal metabolism of keeping cell.Under normal circumstances, cytolemma has the saturating sexuality of selection to material.When plant was subject to adverse circumstance and affects, cytolemma was destroyed, and membrane permeability increases, thereby makes intracellular Electrolyte Leakage, so that the specific conductivity of vegetable cell vat liquor increases.The degree that membrane permeability increases is relevant with environment stress intensity, also relevant with the power of stress resistance of plant.Like this, same crop different varieties gets final product a little less than the strong stress resistance between control variety in identical increase degree of coercing membrane permeability under the temperature.1991, Mckay pointed out to represent with relative conductivity the size of cell leakage, the size of cell electrolyte leakage rate namely, and it can reflect plant cell membrane saturating property variation and damaged membrane degree of hindering under various adverse environmental factors.Therefore, conductometric titration has become a weak accurate and practical method of plant identification strong stress resistance in crop resistance cultivation, the breeding at present.In plant stress-resistance research, it is generally acknowledged the strong plant variety of salt resistance ability under salt is coerced, cell leakage changes less, and sensitive plant then changes greatly.

Table 20 240mM NaCl coerces the difference unit of lower plant leaf relative conductivity: (%)

Can find out from table 20, Figure 23, during 240mM NaCl salt side of body 24h-14d, the chlorophyll content integral body of wild-type plant is in rising trend, and transgenosis plant during 240mM NaCl salt side of body 24h-48h, the blade relative conductivity is reduction trend, then bottom out after the 72h, but the blade relative conductivity in the time of 14 days is starkly lower than the wild-type plant; Blade relative conductivity analytical results shows: under 240mM NaCl condition of salt stress, the cytolemma of wild-type plant is subject to the impact of high salt, degree of injury is larger, and the transfer-gen plant of insertion BgERF transcription factor, can produce acknowledgement mechanism at the environment stress initial stage, alleviated the damage of high salt cell membrane, thereby the assurance plant can be kept the normal growth growth in high salt adverse circumstance.

Turning the BgERF gene plant can identify from two aspects the resistance of high salt: the first, and the morphological specificity under the high-salt stress adverse circumstance, the second, the variation of plant and degeneration-resistant relevant every physical signs under the high-salt stress adverse circumstance.The aspects such as the plant height of the present transfer-gen plant of change list of its morphological specificity, blade size, setting percentage; Aspect physical signs: soluble sugar is as osmotic adjustment, accumulation under osmotic stress, help vegetable cell to reduce osmotic potential and the flow of water, reduce osmotic stress to the harm of plant, the content of these osmotic adjustments also becomes the important indicator of Osmotic Stress Tolerance In Plant; And mda (MDA) is the final degradation production of peroxidation of membrane lipids, and its content can reflect that plant suffers the degree of adverse circumstance injury.Indirectly weigh the photosynthesis of plant under high-salt stress with measuring chlorophyll content; The blade specific conductivity is the same with mda also to be that the high salt adverse circumstance of reflection is to the extent of injury of plant cell membrane.This examination example is studied wild-type plant and transgenosis type plant to the tolerance of salt stress by measuring wild-type and the transgenosis type plant every physical signs under the different time salt stress is processed.The result shows: the plant leaf water content of transfer-gen plant, chlorophyll content are higher than contrast, and blade relative conductivity, mda and soluble sugar content all are lower than contrast, this five indices all becomes the important biochemical evidence that the transgenic plant saline-alkaline tolerance improves, thereby also further specify the salt tolerance that the BgERF gene may improve by the content of regulating osmotic adjustment transgene tobacco, to keep the normal biochemical functions of plant.

The above only is the preferred embodiments of the present invention, only is illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skills understand, and can carry out many changes to it in the spirit and scope that claim of the present invention limits, revise, even the equivalence change, but all will fall within the scope of protection of the present invention.

Claims (9)

1. Bruguiera conjugata (Bruguiera gymnorhiza (L.) Lam) ERF transcription factor, it is characterized in that: described transcription factor contains the aminoacid sequence shown in the SEQ ID NO:3.

2. the cDNA sequence of the Bruguiera conjugata ERF transcription factor claimed in claim 1 of encoding.

3. according to cDNA sequence claimed in claim 2, it is characterized in that: described cDNA sequence be shown in the SEQ IDNO:1 or SEQ ID NO.2 shown in.

4. the plant expression vector that contains claim 2 or 3 described cDNA sequences.

5. the Host Strains that contains the described plant expression vector of claim 4.

6. the application of Bruguiera conjugata ERF transcription factor claimed in claim 1 in improving stress resistance of plant or seed selection resistance crop.

7. claim 2 or the 3 described cDNA sequences application in improving stress resistance of plant or seed selection resistance crop.

8. according to application claimed in claim 7, it is characterized in that, comprising: make up the plant expression vector that contains claim 2 or 3 described cDNA; Constructed plant expression vector is transformed in plant or the vegetable cell, cultivates the transgenic plant that obtain the tolerance to environmental stress raising.

9. according to application claimed in claim 7, it is characterized in that: described resistance comprises that salt tolerant is coerced, drought-resistant or anti-low temperature.

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