CN103319584B - 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 cDNA sequence and expression vector thereof and application

Technical field

The present invention relates to the transcription factor of a kind of cDNA sequence relevant to stress resistance of plant and coding thereof, the cDNA sequence of coding ERF transcription particularly relating to from mangrove plant Bruguiera conjugata (Bruguiera gymnorhiza (L.) Lam) institute and be separated, clone, the invention still further relates to high efficiency plant expression vector containing this cDNA sequence and they are improving the application in stress resistance of plant, belonging to plant genetic engineering field.

Background technology

Saline and alkaline is the main abiotic stress factor affecting terrestrial plant growth and restriction crop yield.According to statistics 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 also has increasing trend.Salinization soil not only occupies very large proportion in China's cultivated area, other various countries are also like this in the world, such as according to the incomplete statistics of WHO (UNESCO) and the World Food Programme (FAO), in world wide 9.6 hundred million square kilometres, namely the land of more than 30% is covered by saline-alkali soil, and in cumulative year after year trend; The irrigation district of whole world half is subject to the impact of Secondary Saline and waterlogging to some extent.In the existing cultivated area of China, 6,700,000 hectares are about had to be subject to saline and alkaline harm at present, and along with process of industrialization quickening, the continuous expansion of irrigated land and plastic greenhouse area, the soil salinization area of China will further expand, this, by seriously restricting the utilization of China's agricultural to 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 has produced agroforestry and has caused huge harm.Therefore, along with arable land in world wide is reduced and being on the rise of salt marsh day by day, in the face of China's population constantly increases, to plough and liquid manure resource reduces day by day, environmental aspect sharply worsens, agriculture production is more and more subject to stern challenge, salt resistance ability how by improving crop increase grain and cash crop output, economize on resources and environmental protect, become the great demand of China's Sustainable Agricultural high-efficient development.And existing farm crop mostly belong to non-halophytic seeds.How to make non-Halo crop can ensure high yield again by normal growth in saline soil environments, this is one of focus of scientists study for a long time.Population increase is faced in the world today, under the severe situation that limited and effective cultivated area constantly reduces, develop large-area saltings, cultivate salt tolerant crop new variety and from then on go on the arena of history, become and solve one of world food and energy starved effective way.

In agriculture production the core content of mankind's activity be constantly improve plant growth environment and constantly more new variety to adapt to changeable ecotope, make the mankind obtain how better product.Along with completing successively of various plants genome blueprint examining order, on a molecular scale plant is carried out to the research of the degeneration-resistant mechanism of coercing, understand the patience mechanism of plant; Excavate and 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 improving farm crop, expand arable area, increase crop yield, will the inexorable trend of the future of agriculture production be become.

Mangrove forest generally includes the shrub of growth in the torrid zone, tideland, tidal swamp, subtropics and subtidal zone shoaling water and arbor, the living environment of swampland tide bulge and fall in sea salt beach or bay has finally been adapted to through very long Historical Evolution, due to be subject to seawater periodically leaching flood and be rich in Weibull, become red by felling rear oxidation, therefore claim " mangrove ".The composition of mangrove forest is based on kind rhizophoraceous, and Rhizophoraceae has 16 genus 120 kinds, and part growth is in inland, and part composition mangrove forest, 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 regional.In China, the tropical and subtropical zone seashore of the silting in the Hainan Island that mangrove forest is mainly distributed in, Guangxi, Guangdong and Fujian and bay, or the alluviation solonchak at river outlet place or saliferous silty loam, be suitable for mangrove forest advolution.

Bruguiera conjugata [Bruguiera gymnorhiza (L.) Lam] belongs to Rhizophoraceae Bruguiera conjugata and belongs to, and is grown on the tideland, river mouth, bay that sea, Perenniporia martius land crosses." arbor or shrub, high 2-4m, has stilit root, leaf keratin and mutual to life, ovalize.Flower both sexes, cyme, fruit obovate is sagging, long 20-40cm.Bruguiera conjugata plays an important role safeguarding in coastal ecology balance, environmental monitoring, water body purification ".They can adapt to, and sea wind corrodes, the living environment of salt water immersion and anoxic substrate, grows green and fresh all the year round.In salt-tolerant plant molecular breeding, preferably go the theory of excavating to Saline Region based on the genetic material improving plant anti-salt performance.We clone the transcription factor gene be closely related with salt tolerance from mangrove Bruguiera conjugata, can not only provide excellent genes source, and seem particularly important and urgent for crop breeding for stress tolerance for genetic engineering breeding.

Plant stress-resistance genetically engineered imports discrete function gene to improve certain resistance, but do not reach and make the resistance of plant obtain improvement that is comprehensive, essence.The resistance of plant is not embodied by expression that is single or a few gene, but by the quantitative character of controlled by multiple genes.Although clone a large amount of anti contravariance related genes successively from each kind of plant at present, these gene great majority can only increase certain single resistance of plant, comprehensively can not improve the resistance of plant on the whole.The by-pass cock that transcription factor is expressed as functional gene, different genes can be regulated accurately, crucial effect is played in plant adverse circumstance signal transduction process, so the effect by strengthening certain transcription factor, multiple and degeneration-resistant relevant functional gene can be impelled to express, and this is the very effective approach making plant stress-resistance proterties obtain comprehensive improvement.ERF (ethylene-responsive element binding factor) class transcription factor is a subfamily of AP2/EREBP transcription factor family, the very conservative DNA binding domain that each member is made up of about 60 amino acid containing.From structural analysis of protein, ERF class transcription factor contains 4 functional domains, i.e. DNA binding domain (DNA-binding domain), transcriptional regulatory domain (transcription regulation domain) (comprising Activation and inhibition territory), oligomerisation site (oligomerization) and nuclear localization signal (nuclear localization signal, NLS).Wherein, the ERF/AP2DNA binding domains of high conservative contains and is made up of 60 ~ 70 amino-acid residues, comprise 3 β-pleated sheet structures and 1 α spiral, by with goal gene upstream promoter region 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.

Found to there is a large amount of transcription factor in plant materials at present, Arabidopis thaliana (A rabidopsis thaliana) only containing 27000 genes, wherein just has the gene of 5.9% to be encoding transcription factors.So far in Arabidopis thaliana find 124 ERF family genes, in paddy rice find 139 ERF family genes, in recent years also from other plant as this kind of family gene isolated by tobacco, tomato, corn etc.Only there is part to carry out functional analysis in the ERF gene of successful clone and report, but not yet met report in Rhizophoraceae plant.

Research shows: the expression of most of ERF gene is all subject to the induction of arid, saline and alkaline, low temperature, freeze injury, the environment stress such as disease and mechanical wounding.There is report Tsil to be done mutually by its ERF binding domain and cis element GCC-box and DRE, improve salt tolerance and the disease resistance of transgene tobacco simultaneously; Degeneration-resistant test proves that 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, create new degeneration-resistant material and provide material base.

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

Summary of the invention

An object of the present invention is to provide a kind of Bruguiera conjugata ERF transcription.

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

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

Two of object of the present invention is to provide the cDNA sequence of described transcription factor of encoding.

The cDNA sequence of a kind of Bruguiera conjugata ERF transcription of encoding of the present invention, its cDNA sequence of coding ERF transcription being from mangrove plant Bruguiera conjugata (Bruguiera gymnorhiza (L.) Lam) institute and being separated, cloning.

In a particular embodiment of the present invention, described cDNA sequence is for SEQ ID NO:1 Suo Shi or shown in SEQ ID NO.2.

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

The present invention utilizes DNAMAN software to carry out homogeneous assays to the Nucleotide of the relevant section platymiscium AP2/ERF genoid announced in Genbank and aminoacid sequence, according to this kind of gene DNA binding domain conserved regions design synthesis pair of degenerate primers, utilize RT-PCR method from mangrove plant Bruguiera conjugata (Bruguiera gymnorhiza (L.) Lam), be separated to the homologous fragment of AP2/ERF genoid, then new AP2/ERF transcription factor gene BgERF (shown in SEQ ID NO:1) is obtained by RACE-PCR technology clone, the cDNA full length sequence using ORF finder software analysis BgERF gene is 1870bp, entire open reading frame 1428bp (262bp-1689bp, comprise initiator codon and terminator codon, as shown in SEQ ID NO.2), to encode 475 amino acid, 5 '-UTR (1bp-261bp), 3 '-UTR (1690bp-1870bp), SMART software is analyzed the protein structure domain that BgERF transcription factor is encoded, and result shows that BgERF albumen contains the typical AP2/EREBP structural domain that is positioned at 241-304 position, is made up of, has YRG and RAYD two Conserved Elements 64 amino acid.FeatureMap3D software analysis is utilized to predict the three-D space structure of BgERF albumen, result shows: have 1 α spiral in the protein steric structural coded by BgERF respectively, 3 β-pleated sheet structure compositions, this is the space structure that typical ERF class transcription factor has.

Three of object of the present invention is to provide a kind of plant expression vector of the cDNA sequence containing described transcription factor and the Host Strains containing 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 object of the present invention is to provide described Bruguiera conjugata ERF transcription and cDNA sequence thereof improving the application in stress resistance of plant or seed selection resistance crop, comprising: build the plant expression vector containing the above cDNA; Constructed plant expression vector is transformed in plant or vegetable cell, cultivates the transgenic plant obtaining improving tolerance to environmental stress.

Wherein, described resistance comprises resistance to salt stress, drought-resistant or anti-low temperature.

By the plant expression vector containing ERF transcription nucleotide sequence of the present invention by Agrobacterium-medialed transformation method transformation of tobacco, to the plant leaf water content of transfer-gen plant, chlorophyll content, specific conductivity and mda content etc. measure, found that the plant leaf water content of transfer-gen plant, chlorophyll content is all higher than contrast, and blade relative conductivity, mda and soluble sugar content are all lower than contrast, this five indices all becomes the important biochemical evidence that transgenic plant saline-alkaline tolerance improves, also further illustrate BgERF gene and pass through to regulate the content of osmotic adjustment thus the salt tolerance improving transgene tobacco, maintain the normal biochemical functions of plant.

Accompanying drawing explanation

Fig. 1 is Bruguiera conjugata Total RNAs extraction figure;

Fig. 2 is degenerate pcr amplification;

M:Marker 1: degenerate pcr result:

Fig. 3 is 3`RACE-PCR amplification;

M:Marker 1:PCR result

Fig. 4 is 5`RACE-PCR amplification;

M:DL2000Marker 1:PCR result

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

M:DNA Marker 1:PCR product

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

Fig. 7 is the Phylogenetic analysis result of BgERF aminoacid sequence;

A. Arabidopis thaliana; B. capsicum; C. the phase radish; D. upland cotton; E. soybean; F. small bell thorn; 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 cuts result;

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

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

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

Figure 16 is the PCR detected result of the DNA level turning BgERF genetic tobacco;

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

Figure 17 is that the PCR of the rna level turning 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 preparation method of the present invention and beneficial effect by the following examples, it should be understood that these embodiments only for the object of illustration, never limit the scope of the invention.

Illustrate: in following examples, do not make the experimental methods of molecular biology illustrated, equal reference " Molecular Cloning: A Laboratory the guide " (third edition, J. Pehanorm Brooker) concrete grammar listed in a book carries out, or operates according to reagent kit product specification sheets.

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

1 materials and methods

1.1 material

1.1.1 vegetable material is cultivated and Stress treatment

Bruguiera conjugata branch is collected in Shenzhen Mangrove woods wilderness area.With the NaCl of 250mM by Bruguiera conjugata spray immersion treatment 5h, win young leaflet tablet and use liquid nitrogen process immediately, be stored in-80 DEG C 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 purchased from American hero's life technology company limited (Invitrogen); DNA glue recovery test kit and high-purity Plasmid Miniprep Kit are all purchased from Beijing hundred Tyke Bioisystech Co., Ltd; Taq enzyme, 3 ' full RACE Kit and 5 ' full RACE Kit is purchased from TaKaRa company; Restriction enzyme, T4 ligase enzyme etc. are purchased from NEB company; First chain cDNA synthetic agent box is purchased from MBI company.Primer synthesis and gene sequencing are completed by Beijing AudioCodes Bioisystech Co., Ltd.

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), the chloro-3-indoles of the bromo-4-of 5--β-D-galactoside (X-gal) etc. are respectively purchased 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 Tris-HCl (pH 8.0,500ml): Tris 60.6g; 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 major experimental operating instrument

PCR amplification instrument (Bio-RAD),-80 DEG C 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, 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 Bruguiera conjugata total serum IgE

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

1.2.2 the synthesis (being undertaken by Invitrogen company Reverse Transcription box specification sheets) of the first chain cDNA

1. denaturation: with Bruguiera conjugata total serum IgE for template take Oligo-dT as primer (working concentration is 10pmol/ μ l).Reaction system, as table 1, mixes rear 70 DEG C of sex change 5min, is placed in 2min on ice immediately;

Table 1RNA denaturation system

2. reverse transcription: mix add following ingredients listed in above-mentioned centrifuge tube after, moment is centrifugal, 42 DEG C of 60min, 70 DEG C of 10min deactivation ThermoScript II termination reactions, and-20 DEG C save backup;

Table 2 reverse transcription system

3.RNaseH digests: the RNaseH adding 1 μ l (2U/ μ l) in reaction solution, 37 DEG C of 20min, digests the single stranded RNA be combined with cDNA ,-20 DEG C 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 and synthesis of primer

DNAMAN software is utilized to carry out homogeneous assays to the Nucleotide of the relevant section platymiscium AP2/ERF genoid announced in Genbank and aminoacid sequence, according to this kind of gene DNA binding domain conserved regions design synthesis pair of degenerate primers, primer synthesizes (see table 3) by Beijing AudioCodes biotechnology limited liability company.

The primer used in table 3 degenerate pcr

1.2.3.2RT-PCR

With mangrove Bruguiera conjugata leaf cDNA for template, use degenerated primer MEF and MER carries out RT-PCR, PCR condition and is: 94 DEG C of 3min, (94 DEG C of 45sec, 55 DEG C of 45sec, 72 DEG C of 45sec) 30cycles, 72 DEG C of 10min.The agarose gel electrophoresis that reaction terminates rear use 1% is identified PCR primer.

1.2.3.3PCR the recovery of product

Adopt the Easypure PCR Purification kit of Beijing Quanshijin Biotechnology Co., Ltd to reclaim object fragment, operate according to the method for test kit specification sheets.

1.2.3.4 the connection of object fragment and cloning vector

The PCR primer of recovery be connected on pMD19-T carrier, ligation system is as table 4, and 16 DEG C of connections are spent the night.

Table 4 ligation system

1.2.3.5 the preparation of E. coli competent

Carry out with reference to concrete grammar listed in " Molecular Cloning: A Laboratory guide " (third edition, J. Pehanorm Brooker) book.

1.2.3.6 colibacillary conversion (heat shock method)

Carry out with reference to concrete grammar listed in " Molecular Cloning: A Laboratory guide " (third edition, J. Pehanorm Brooker) book.

1.2.3.7 bacterium colony PCR identifies

With the white list bacterium colony of picking on the LB flat board containing Amp, carry out bacterium colony and cultivate and PCR checking, primer is MEF and MER, and amplification condition is: 94 DEG C of 3min, (94 DEG C of 45sec, 55 DEG C of 45sec, 72 DEG C of 40sec) 30 circulation, 72 DEG C of 10min.The agarose gel electrophoresis that reaction terminates rear use 1% is identified PCR primer, and screening is obtained positive colony and 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 and synthesis of primer

According to the homologous fragment of acquired ERF gene, the nested gene special primer of design two to 3 ' RACE, is synthesized, in table 5 by Beijing AudioCodes biotechnology limited liability company.

The primer used during table 53 ' RACE reacts

1.2.4.2 the extraction of Bruguiera conjugata total serum IgE

With Bruguiera conjugata blade for material adopts TRIzol method to extract total serum IgE.

1.2.4.3 the synthesis of the first chain cDNA

The same 1.2.2 of method.

1.2.4.4 nested PCR amplification 3 '-end cDNA

Take cDNA as template, gene specific primer ME3F1 and ME3F2 and 3 ' RACE reverse primer 3-Outer and 3-Inner is used to carry out 50 μ l system two-wheeled pcr amplifications respectively, reaction parameter is: 94 DEG C of 3min, (94 DEG C of 45sec, 56 DEG C of 45sec, 72 DEG C of 60sec) 30cycles, 72 DEG C of 10min.First round PCR primer is diluted 100 times and takes turns amplification template as second, finally amplified production 2% agarose gel electrophoresis is detected, reclaim PCR primer and transformation of E. coli DH5 α, check order after utilizing α complementation and bacterium colony PCR screening positive clone.

1.2.5 nested PCR amplification 5 ' holds cDNA

1.2.5.1 the Design and synthesis of primer

Design two pairs of nested gene special primers according to acquired 3 ' end cDNA sequence, by Beijing AudioCodes, biological skill limited liability company synthesizes (as shown in table 6).

The primer used during table 65 ' RACE reacts

1.2.5.2 the synthesis of the first chain cDNA

Use TaKaRa 5 ' Full RACE Kit to carry out CIAP, TAP process to Bruguiera conjugata blade Total RNA, and after being connected with 5 ' RACEAdaptor, reverse transcription synthesis cDNA, set up M-MLV (-) to contrast simultaneously.(method illustrates see TaKaRa 5 ' Full RACE Kit and Invitrogen company Gene RacerTM kit test kit)

1.2.5.3 nested PCR amplification 5 ' holds cDNA

The cDNA obtained with reverse transcription is for template, and use 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 respectively, reaction system is in table 7 and table 8.Two-wheeled PCR reaction conditions is: 94 DEG C of 3min, (94 DEG C of 45sec, 57 DEG C of 45sec, 72 DEG C of 2min) 30cycles, 72 DEG C of 10min.The agarose gel electrophoresis of 0.8%, reclaims PCR primer and is connected to pMD19-T carrier, transformation of E. coli DH5 α, utilizes α complementation and bacterium colony PCR screening positive clone, and send AudioCodes order-checking portion to check order, 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 increases

1.2.6.1 the Design and synthesis of primer

Splice known 3 '-end and 5 '-end cDNA sequence, obtain cDNA full length sequence, accordingly, design gene specific primer, the cDNA full length sequence of amplification Bruguiera conjugata ERF gene, the primer is in table 9.

1.2.6.2 the synthesis of the first chain cDNA

The same 1.2.2 of method.

Table 9cDNA full length sequence increases the primer used

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

1. obtain cDNA for template with reverse transcription, utilize gene specific primer MERFF and MERFR, carry out pcr amplification, reaction parameter: 94 DEG C of 5min; 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 2min, 30 circulations; 72 DEG C of 10min.

2.0.8% agarose gel electrophoresis, reclaim PCR primer and be connected to pMD19-T carrier, transformation of E. coli DH5 α, 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; Online ORF finder software analysis http://www.ncbi.nlm.nih.gov/gorf/orfig.cg is utilized to carry out 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; Online ExPASy Compute pI/Mw tool software http://www.expasy.ch/tools/pi_tool.html is utilized BgERF albumen to be carried out to the prediction of physical properties; FeatureMap3D software analysis is utilized to predict the three-D space structure of BgERF albumen; NCBI-BLAST and DNAMAN software is utilized 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 is all work in nucleus usually.BgERF has the nuclear localization sequence of prediction.The prompting of this constructional feature they may be positioned at nucleus and work as core transcription modulator.This test builds green fluorescent protein (green fluorescent protein, GFP) fusion expression vector p163-BgERF-GFP, with empty carrier p163-GFP for contrast, Bombardment-Mediated Transformation onion epidermis cell, laser scanning co-focusing microscope observes the Subcellular Localization situation of checking BgERF albumen.

1. design of primers and synthesis

Design and synthesis contains primer GFPHM-SalI (F) and the GFPHM-BamHI (R) of restriction enzyme site SalI and BamHI respectively, and sequence is in table 10.

Table 10 Subcellular Localization the primer

The structure of 2.GFP fusion expression vector

Primer GFPHM-SalI (F) and GFPHM-BamHI (R) is utilized to amplify complete open reading frame (shown in SEQ ID NO.2 from the pMD19-T carrier being connected with total length BgERF cDNA.)。Amplified production and p163-GFP carrier are after double digestion, agarose gel electrophoresis, recovery, and BgERF is connected in SalI and the BamHI site of p163-GFP carrier by T4 ligase enzyme, build and translate with GFP the expression vector p163-BgERF-GFP merged.

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 is extracted (alkaline process) and enzyme and is cut qualification

Method cuts explanation with reference to hundred Tyke Plasmid Miniprep Kit and TaKaRa company enzyme, and enzyme cuts system in table 11.

Table 11 endonuclease reaction system

5. Bombardment-Mediated Transformation onion epidermis cell

5.1 materials prepare

Onion entocuticle (layer 5) is cut into 0.5cm × 0.5cm fritter, and back of tearing faces up, and puts in MS substratum, preculture 4h.

5.2 bronze suspension preparations

1) take 20mg (40 rifle) 1.0 μm of ol/L bronzes, put into 1.5ml centrifuge tube, add 500 μ l dehydrated alcohols, the centrifugal 1min of vortex oscillation 1min, 10000r/min, abandons supernatant, repeats 3 times;

2) add the distilled water of 500 μ l sterilizings, the centrifugal 1min of vortex oscillation 1min, 10000r/min, abandons supernatant, repeats 2 times;

3) add the distilled water of 400 μ l sterilizings, vortex oscillation suspension bronze, under turbula shaker at a slow speed non-stop-machine situation, bronze is evenly distributed in 10 0.5ml centrifuge tubes, often pipe 40 μ l, 4 DEG C of preservations;

5.3 particle gun particulate bullet preparations

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) mixing vibration mixing 3min;

3) 15min is left standstill on ice;

4) the centrifugal 10sec of 12000r/m (referring to that rotating speed reaches 12000 rear 10sec);

5) abandon supernatant, add 140 μ l dehydrated alcohols slightly vibrate after (breaing up bronze), the centrifugal 10sec of 12000r/m collects bronze precipitation;

6) 20 μ l dehydrated alcohols suspension precipitations are added, stand-by.

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

5.5Leica TCS SP2 confocal laser scanning microscope result

1.2.7.2BgERF the checking of transcription factor transcriptional activation function

Build pBridge-BgERF yeast effect plasmid, proceed in yeast strain AH109, and respectively the mono-defect substratum of the SD lacking tryptophane and lack tryptophane, Histidine the two defect of SD SD substratum on screen positive transformant, carry out Whatman filter paper colour developing qualification and betagalactosidase activity simultaneously and detect and nextly tentatively by these two transcription factors of yeast one-hybrid verification experimental verification, there is transcriptional activation function.

1. the Design and synthesis of primer

Design and synthesis primer MEZ (F) and MEZ (R), sequence is in table 12.

The primer used in table 12 yeast expression

2. the structure of effect plasmid

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

3. conversion 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 is extracted (alkaline process) and enzyme and is cut the plasmid of qualification through detected through gel electrophoresis and carry out the reaction of BamHI and SalI double digestion, and endonuclease reaction system is with table 13.

Table 13 endonuclease reaction system

5. the preparation of competent yeast cells and conversion

With reference to lithium acetate transformation method listed 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 measure

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

The acquisition of 2.1ERF DNA homolog fragment

Homology compare of analysis has been carried out to the ERF protein sequence reported, find that the aminoacid sequence of the DNA binding domain of ERF albumen all presents the characteristic of high conservative in different plant, different ERF family, therefore the design of degenerated primer that utilized this conservative region to carry out, with the blade total serum IgE of salt stress process, (the RNA electrophoresis result of extraction as shown in Figure 1.) cDNA that obtains of reverse transcription is template, utilizes the degenerated primer of design to go out the object tape (Fig. 2 shown in) of about 200bp-300bp by pcr amplification.The PCR primer of recovery is connected on pMD19-T carrier, transformation of E. coli DH5 α, utilizes α complementation and bacterium colony PCR to filter out positive colony, after order-checking, obtain the middle conserved sequence of 262bp.Shown 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, tentatively judges that this fragment is Halimodendron halodendron ERF gene conserved regions.

The acquisition of 2.2 Bruguiera conjugata ERF gene cDNA 3 ' sequences 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 two-wheeled pcr amplification, electrophoresis detection obtains the amplified production (shown in Fig. 3) of about 700bp, sequencing result shows that this fragment length is 639bp, and 5 ' terminal sequence has 86bp overlapping with former homologous fragment respectively.

By the homologous fragment that obtained and 3 ' end fragment sequence, 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 the amplified production (shown in Fig. 4) of two treaty 1000bp and 500bp respectively.

Reclaim this two specific fragments respectively, the multiple positive transformants cloning and sequencing of picking, result shows that the sequence that 1000bp sequencing fragment obtains is the correct fragment of 5 ' RACE, and sequencing result is 969bp, and 3 ' terminal sequence has 91bp overlapping with former homologous fragment respectively.And experimental design M-MLV (-) contrasts.

The acquisition of 2.3 Bruguiera conjugata ERF gene cDNA full length sequences

5 ' end and 3 ' are held and homologous sequence splicing, design full length gene special primer MERFF and MERFR, carries out pcr amplification, obtains about 1000-2000bp product band (shown in Fig. 5).Sequencing result shows, this full length sequence is 1870bp, and the display of sequence alignment result has higher similarity with the gene of various plants ERF family, and be inferred as new ERF gene, called after BgERF, its nucleotide sequence is as shown in SEQ ID NO.1.

2.4BgERFcDNA full length sequence is analyzed

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

The cDNA full length sequence using ORF finder software analysis BgERF gene is 1870bp, entire open reading frame 1428bp (262bp-1689bp), to encode 475 amino acid, 5 '-UTR (1bp-261bp), 3 '-UTR (1690bp-1870bp); SMART software is analyzed the protein structure domain that BgERF transcription factor is encoded, and result shows that BgERF albumen contains the typical AP2/EREBP structural domain that is positioned at 241-304 position, is made up of, has YRG and RAYD two Conserved Elements 64 amino acid.YRG is conducive to the combination with DNA, RAYD can by affect YRG district conformation or by regulating the combination of AP2 structural domain and DNA with the interaction of other albumen.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 prediction of albumen

FeatureMap3D software analysis is utilized to predict the three-D space structure (shown in Fig. 6) of BgERF albumen, as figure shows: have 1 α spiral in the protein steric structural coded by BgERF respectively, 3 β-pleated sheet structure compositions, 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 Phylogenetic analysis

AP2/ERF family clone gene mRNA sequence is searched in NCBI, carry out gene order comparison and Phylogenetic analysis with DNAMAN software, result display most crop ERF class transcription factor conservative district such as Bruguiera conjugata and paddy rice, upland cotton, capsicum, tomato, soybean, Radix Dauci Sativae, Arabidopis thaliana, potato has higher homology.Sequence alignment result (as shown in Figure 7) shows: BgERF gene is together with the crop such as Arabidopis thaliana, paddy rice cluster, and tomato, Radix Dauci Sativae, upland cotton, soybean, small bell thorn cluster together, thus same gene acid molecules is evolved and still be there is certain difference in visible different plant species, can understand by the comparison of sequence homology and Phylogenetic analysis the inherent law that the evolution of gene and biosystem occur.

The Subcellular Localization of 2.5BgERF transcription factor

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

Vector construction schema as shown in Figure 8.

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

The checking of 2.6BgERF transcription factor transcriptional activation function

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

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: result as shown in figure 11.

2.6.2.2BgERF the betagalactosidase activity analytical results of albumen in yeast: 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 correlation function checking 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, this laboratory is preserved; Dicotyledons expression vector pBI121 (illustrating: pBI121 is that plant expression vector is commonly used in laboratory, and NCBI can find its complete sequence and explanation thereof), this laboratory is preserved.PMD19-T cloning vector, purchased from TaKaRa.

1.1.3 reagent

Various restriction enzyme, rTaq enzyme, ExTaq enzyme are purchased from Takara company; T ₄dNA ligase is purchased from Invitrogen company; Taq enzyme is purchased from Beijing Quanshijin Biotechnology Co., Ltd; Gel/PCR Extraction Kit is purchased from BioMIGA company.

1.1.4 substratum

1.YEB substratum:

With reference to concrete grammar listed in " Molecular Cloning: A Laboratory guide " (third edition) J. Pehanorm Brooker one book;

2.LB substratum:

With reference to concrete grammar listed in " Molecular Cloning: A Laboratory guide " (third edition) J. Pehanorm Brooker one book;

3.MS substratum:

Concrete grammar listed by editing see practical plant tissue culture technique study course (Gansu science tech publishing house) Cao Ziyi;

4.MS Selective agar medium:

6-BA (3.0mg/L) is added, NAA (0.2mg/L), Cephradine (500mg/L) and kantlex (100mg/L) in MS minimum medium

5.MS root media:

Cephradine (500mg/L) and kantlex (200mg/L) is added in MS minimum medium

1.2 experimental technique

1.2.1 design of primers and synthesis

Design and synthesis contains primer pBI121-XbaI (F) and the pBI121-SacI (R) of restriction enzyme site XbaI and SacI.Sequence is in table 15.

Table 15 builds 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

Primer BgERF-XbaI (F) and BgERF-SacI (R) is utilized to amplify complete open reading frame (shown in SEQ ID NO.2 from the pMD19-T carrier being connected with total length BgERF cDNA.)。Amplified production and carrier after double digestion, agarose gel electrophoresis, recovery, T ₄bgERF is connected to XbaI and the SacI site of carrier pBI121 by ligase enzyme, replace the GusA gene on carrier pBI121, build plant expression vector pBI121-BgERF, transformation of E. coli DH5 α, pcr amplification screening recombinant plasmid, and carry out corresponding enzyme and cut qualification;

1.2.2.2 recombinant plasmid a small amount of extract and enzyme cut qualification

Plasmid extraction method is with reference to the concrete grammar listed by hundred Tyke Plasmid Miniprep Kit; Enzyme is cut qualification 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 are see the preparation of fine works molecular biology experiment guide (Ao Sibai) single stage method and transformed competence colibacillus cell.

The PCR qualification of the Agrobacterium 1.2.3.2 containing recombinant plasmid

Picking 28 DEG C cultivates the YEB of 1-2d (containing 50mg/L Kan respectively, 50mg/L Rif) single bacterium colony on transformation plate, make negative control with unconverted Agrobacterium, with plant expression carrier plasmid pBI121-35S-BgERF for positive control, carry out bacterium colony PCR qualification.

1.2.4 the detection of BgERF genetic tobacco is turned

1.2.4.1DNA the PCR of level detects

Utilize CTAB method to extract the tobacco gene group DNA containing Kan resistance in a small amount, detect the positive tobacco plant of screening further by PCR.If amplified target fragment, and do not amplified fragment in negative control, namely preliminary proof goal gene has been incorporated in 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 serum IgE, reverse transcription obtains cDNA as template, and RT-PCR detects screening positive transformants seedling.1.2.1-1.2.2 in method reference embodiment 1, PCR reaction parameter: 94 DEG C of 5min; 94 DEG C of 30sec, 60 DEG C of 30sec, 72 DEG C of 2min, 30 circulations; 72 DEG C of 10min; The agarose gel electrophoresis of 0.8%.

1.2.5 the resistance to of transgene tobacco coerces analysis

1.2.5.1 the abiotic stress resistance qualification of transgene tobacco

High-salt stress Resistance Identification is carried out, if three repetitions with the transplanting growth tobacco plant of about 2 weeks:

Tolerant salt: water by the NaCl solution of 0mM, 120mM, 240mM respectively and have in the basin of wild-type and transgenic tobacco in cultivation, process 2 weeks in 22 DEG C of illumination boxs; Respectively in 0h, 24h, 48h, 72h, 14d sampling, after liquid nitrogen process, be placed in-80 DEG C of preservations;

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

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

To transgenosis and the non-transgenic tobacco of different concns Nacl Stress treatment, get blade respectively at 0h, 24h, 48h, 72h, 14d, clean with distilled water flushing, thieving paper blots surface-moisture.3 individual plants are got in each process, and survey its fresh weight rapidly, and put into 120 DEG C of baking ovens afterwards and toast 20min, 80 DEG C of bakings are spent the night to constant weight, weigh its dry weight.To above-mentioned dry, the fresh weight recorded, 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 the tobacco leaf 0.5g through Stress treatment, liquid nitrogen freezing is also with after historrhexis's instrument fragmentation, add 1ml0.5%TBA, after vortex concussion homogenate, again above-mentioned homogenate is transferred to clean 10mlEp manage and number (contrast adds 1ml distilled water), and then add 0.5%TBA solution 5ml, boil after boiling reaction 15min to sufficient reacting after mixing in a water bath; Put into rapidly ice and cool 10min, draw supernatant low-speed centrifugal; Then the absorbance of 532nm, 600nm and 450nm wavelength place supernatant liquor is surveyed respectively 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)

Note: in formula, A450, A532, A600 are respectively the absorbance of liquid under 450nm, 532nm, 600nm wavelength recorded.

1.2.6.3 transfer-gen plant chlorophyll content is analyzed

Take the tobacco leaf 0.2g through Stress treatment, liquid nitrogen freezing is with after historrhexis's instrument fragmentation, add the acetone soln of 1ml 80%, vortex concussion homogenate, more above-mentioned homogenate is transferred to clean 10mlEp and manages and number (contrast adds 1ml distilled water), and then add the acetone soln of 5ml80%, react 30min until fragment of tissue bleaching, 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

(note: C in formula _t, Ca, Cb unit be mg/L)

1.2.6.4 rotaring gene plant blade organizes relative conductivity analysis

Take tobacco leaf 0.2g through Stress treatment in 10mlEP pipe, add 5ml deionized water, natural immersion 1h, measure specific conductivity J1 afterwards and record, then put into boiling water bath and boil 10min, be cooled to room temperature and survey specific conductivity J2 and record.

The calculation formula of plant tissue relative conductivity:

Relative conductivity=(J1-J0)/(J2-J0) * 100%

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

2 experimental results and analysis

The structure of 2.1 plant expression vectors

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

2.1.2 the bacterium colony PCR of recombinant plasmid identifies

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

2.1.3 the enzyme of recombinant plasmid cuts qualification

The positive bacterium colony that bacterium colony PCR detects is proceeded to LB (Kan+) liquid nutrient medium, and 37 DEG C are shaken bacterium and spend the night, and extract recombinant plasmid, carry out enzyme and cut qualification, recombinant plasmid pBI121-BgERF cuts out target stripe, and enzyme cuts result correctly, sees Figure 14.

The 2.2 Agrobacterium PCR containing expression vector detect

By recombinant plasmid pBI121-BgERF transformation Agrobacterium LBA4404, make template with the bacterium liquid of the Agrobacterium after transforming, unconverted Agrobacterium makes negative control, and plasmid does positive control, carries out bacterium colony PCR qualification.Detected result shows, this recombinant plasmid successfully proceeds in Agrobacterium, sees Figure 15.

The detection of 2.3 turns of BgERF genetic tobaccos

2.3.1DNA the PCR of level detects

Choose Kan resistance seedling, its blade of clip, adopt CTAB method to extract its DNA, with tobacco gene group DNA for template, with pBI121-XbaI (F) and pBI121-SacI (R) for primer, adopt round pcr, Kan resistance seedling is carried out to the detection of DNA level.Result shows, in kan resistance seedling 22 strain of detection, 16 strains are positive, positive rate about 73%, and plant PCR detects and sees Figure 16, proves that BgERF has been incorporated in tobacco gene group.

2.3.2RNA the PCR of level detects

Choosing the positive tobacco plant blades of 16 strains that DNA level identifies respectively is material, extracts total serum IgE, and the cDNA obtained with reverse transcription is for template, and RT-PCR does further to detect to positive seedling.Result shows, 16 strains are in the plant leaf of genomic dna-pcr test positive, and 12 strain test positive, positive rate 75%, plant RT-PCR detects and sees Figure 17.

The resistance to of 2.4 transgene tobaccos coerces analysis

Salt Tolerance Analysis result: the NaCl solution process of transfer-gen plant and WT lines 240mM, WT lines 2 strain yellow leaf after 50 days, stem stalk withers here to lodge and is tending towards dead, and surplus 1 strain survival is bloomed, and turns BgERF gene plant and all survive, 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 BgERF gene plant improves tobacco.

The Determination of Physiological And Biochemical Indices of 2.5 transgene tobaccos under condition of salt stress

2.5.1 salt stress process is on the impact of plant leaf water content

Plant tissue water content is the common counter representing plant tissue water regime.For the tissue of normal growth, the number of water content directly affects the upgrowth situation of plant.There are some researches show, high salt adverse circumstance all can cause the osmotic stress of plant materials, causes cell dehydration, and serious caused cell turgor is lost, and causes death.

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

This example by measuring the change of wild-type and transgenic plant parts water content under stress conditions, thus obtains the direct evidence that transfer-gen plant is significantly increased compared with WT lines salt tolerance in individual level.Result shows, and after high salt (table 16, Figure 19) process, the water content of wild-type and transfer-gen plant is all on a declining curve.Before Stress treatment, the water content of transfer-gen plant and the water content of WT lines do not have notable difference; During 240mM NaCl Stress treatment 0-72h, the leaf water content of transgenic plant parts is starkly lower than the leaf water content of WT lines, when coercing 72h-14 days, the leaf water content of WT lines is reduced to 68.99%, obviously be subject to the impact of salt stress, and the leaf water content kept in balance of transgenic plant parts, still maintain about 89%, this shows the foreign gene BgERF inserted, there is certain mitigation to the cell dehydration under high-salt stress, thus improve the salt tolerance of transgene tobacco under stress conditions.

2.5.2 salt stress process is on the impact of plant MDA content

Plant organ is old and feeble or when injured by environment stress, often peroxidation of membrane lipids occurs, mda (MDA) is the final degradation production of peroxidation of membrane lipids, and its content can reflect the degree that plant is injured by adverse circumstance; Its generation can also aggravate the damage of film.Therefore, mda produce quantity number can represent the degree of Lipid peroxidation metabolism, also indirectly can 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: μm ol/g) of lower plant leaf MDA content

As can be seen from table 17, Figure 20, wild-type type plant is after the process of 240mM NaCl 48h salt stress, just the MDA content of WT lines can be significantly improved, and transfer-gen plant coerces the increase of 0-14d period MDA content slowly at 240mM NaCl, when coercing 14 days, WT lines MDA content is about 1.5 times of transfer-gen plant.The MDA content analysis result of wild-type and transgenic plant parts shows, BgERF transcription factor can reduce lipid peroxdation effect thus ensure the integrity of adipose membrane in cell under environment stress.This may be due to BgERF downstream signal and peroxylradicals resistance molecule (as gsh, catalase, peroxidase etc.) phase coupling, thus protects cytolemma.

2.5.3 the change of plant soluble sugar content after salt stress process

In the environment of coercing, plant is uneven in order to slow down by coercing the physiological metabolism caused, a large amount of some small molecular organic compounds of accumulation in cell.Soluble sugar is one of small molecules solute of stress-inducing, reduces the flow of water by osmoregulation, maintains higher osmotic pressure, ensures the normal physiological function of cell.

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

As can be seen from table 18, Figure 21, when 240mM NaCl salt side of body 0-72h, in plant, soluble sugar content reduces, and may be that its photosynthesis is subject to very large suppression due under the salt stress of high density, and glucose synthesis reduces, and causes soluble sugar content to reduce.Plant is being coerced between 72h-14d, soluble sugar content in body is with regard to bottom out, and the increase of nontransgenic plants transfer-gen plant soluble sugar content is higher than the increase (p < 0.05) of WT lines soluble sugar content.The soluble sugar content analytical results of plant shows, under environment stress condition, plant can spontaneous generation soluble sugar thus ensure normal metabolic process in cell paste.Insert the transfer-gen plant of BgERF transcription factor, acknowledgement mechanism can be produced under adverse environmental factor, do not need to produce too many soluble sugar to resist environment stress.

2.5.4 plant leaf chlorophyll content change after salt stress process

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

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

As can be seen from table 19, Figure 22, when 240mM NaCl salt side of body 0-72h, the chlorophyll content of wild-type and transfer-gen plant is in reduction trend before comparatively coercing; And after coercing 14d, the chlorophyll content of transfer-gen plant returns to front level of coercing; And the chlorophyll content of WT lines still holds downtrending.Can showing by chlorophyll content analytical results of plant, 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 insert the transfer-gen plant of BgERF transcription factor, acknowledgement mechanism can be produced at the environment stress initial stage, thus ensure the normal photosynthesis of plant strain growth.

2.5.5 the change of plant leaf relative conductivity after salt stress process.

Plant cell membrane plays an important role to the microenvironment and normal metabolism that maintain cell.Under normal circumstances, cytolemma has selection permeability ability to material.When plant be subject to adverse circumstance affect time, cytolemma is destroyed, and membrane permeability increases, thus 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 in identical increase degree of coercing membrane permeability at temperature, can resistance between control variety strong and weak.1991, Mckay pointed out the size that can represent cell leakage with relative conductivity, the namely size of cell electrolyte osmotic rate, and it can reflect the degree of plant cell membrane permeability change and damaged membrane wound under various adverse environmental factor.Therefore, conductometric titration become crop resistance cultivation at present, one of the plant identification resistance power accurate method of practicality in breeding.In plant stress-resistance research, it is generally acknowledged that plant variety that salt resistance ability is strong is under salt is coerced, cell leakage change is less, and sensitive plant then changes greatly.

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

As can be seen from table 20, Figure 23, during 240mM NaCl salt side of body 24h-14d, the chlorophyll content entirety of WT lines is in rising trend, and transgenosis plant 240mM NaCl salt the side of body 24h-48h during, blade relative conductivity is reduction trend, then bottom out after 72h, but the blade relative conductivity 14 days time is starkly lower than WT lines; 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 insert the transfer-gen plant of BgERF transcription factor, acknowledgement mechanism can be produced at the environment stress initial stage, alleviate the damage of high salt cell membrane, thus ensure that plant can maintain normal growth and grow in high salt adverse circumstance.

Turn BgERF gene plant to identify from two aspects the resistance of high salt: the first, the morphological specificity under high-salt stress adverse circumstance, the second, the change of plant and degeneration-resistant relevant every physical signs under high-salt stress adverse circumstance.The aspect such as plant height, leaf blade size, setting percentage of the present transfer-gen plant of change list of its morphological specificity; In physical signs: soluble sugar is as osmotic adjustment, accumulation under osmotic stress, contribute to vegetable cell and 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, its content can reflect the degree that plant is injured by adverse circumstance.The photosynthesis of indirect measure plant under high-salt stress is carried out with measurement chlorophyll content; Blade specific conductivity is the same with mda is also that the high salt adverse circumstance of reflection is to the extent of injury of plant cell membrane.This examination example, by measuring wild-type and the every physical signs of transgenic plant parts under the process of different time salt stress, studies WT lines and transgenic plant parts to the tolerance of salt stress.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 are all lower than contrast, this five indices all becomes the important biochemical evidence that transgenic plant saline-alkaline tolerance improves, also further illustrating BgERF gene can by regulating the content of osmotic adjustment thus improving the salt tolerance of transgene tobacco, to maintain the normal biochemical functions of plant.

The foregoing is only the preferred embodiments of the present invention, is only illustrative for the purpose of the present invention, and nonrestrictive; Those of ordinary skill in the art understand, and can carry out many changes in the spirit and scope that the claims in the present invention limit to it, amendment, and even equivalence is changed, but all will fall within the scope of protection of the present invention.

Claims (8)

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

2. the cDNA sequence of Bruguiera conjugata ERF transcription according to claim 1 of encoding.

3. according to cDNA sequence according to claim 2, it is characterized in that: described cDNA sequence is for SEQ ID NO:1 Suo Shi or shown in SEQ ID NO.2.

4. the plant expression vector containing cDNA sequence described in Claims 2 or 3.

5. the Host Strains containing plant expression vector described in claim 4.

6. Bruguiera conjugata ERF transcription according to claim 1 is improving the application in Tobacco Salt or seed selection salt tolerance tobacco.

7. the cDNA sequence described in Claims 2 or 3 is improving the application in Tobacco Salt or seed selection salt tolerance tobacco.

8. according to application according to claim 7, it is characterized in that, comprising: build the plant expression vector containing cDNA described in Claims 2 or 3; Constructed plant expression vector is transformed in tobacco or tobacco cell, cultivates the transgene tobacco obtaining salt tolerance and improve.