CN103173464B - FvMYB1 gene for regulating and controlling salt resistance of fluffy white wax and application thereof - Google Patents

Abstract

The invention relates to a FvMYB1 gene for regulating and controlling the salt resistance of a fluffy white wax and an application thereof, and belongs to the technical field of molecular biology. The nucleotide sequence of the FvMYB1 gene is shown as SEQ ID No.1. Plant materials are converted by constructing excess expression vectors, which indicates that the salt resistance of a plant can be improved by utilizing the excess expression of the FvMYB1 gene. Therefore, the theoretical basis and the gene source for the research on the abiotic stress of the white wax are provided.

Description

A kind of regulate and control FvMYB1 gene and the application thereof of Fraxinus velutina salt tolerance

Technical field

The present invention relates to a kind of regulate and control FvMYB1 gene and the application thereof of Fraxinus velutina salt tolerance, belong to technical field of molecular biology.

Background technology

Forestry has the huge ecological value, but variant to environmental requirement.In recent years along with the utilization of Molecular Marker Assisted Selection Technology and the rise of genetically engineered research, for improvement forest is laid a good foundation.But the at present excavation of pest-resistant, drought-resistant, the Salt And Alkali Tolerance key function gene of forest with utilize the clone, the transgenic breeding that lag behind, carry out molecular mark, functional gene, the research of carrying out the aspect such as creation of forest new germ plasm becomes inexorable trend.

Fraxinus velutina is the important composition member of Oleaceae, is one of important in the world trees.Because Fraxinus velutina has drought-resistantly and saline and alkaline, be widely cultivated in each area.Resistance to Fraxinus velutina has salt tolerant, attractive in appearance, the feature such as wide distributes, but the domestic aspects such as Chinese wax seedling-wood breeding, afforestation technology that the research of Chinese wax is mainly laid particular emphasis on, screening and the molecular mechanism research of the elite plant strain that resistance is stronger are little, and available adversity gene still less.Therefore, excavate the important gene of Fraxinus velutina and there is important researching value for genetic improvement.

Plant is often subjected to coercing of many biologies and abiotic factor in growth and development process, in degeneration-resistant reaction process, plant regulates and controls the expression of correlation function gene in body effectively by its signal transduction path, and then cause a series of physiology, biochemical reaction, form the signals-modulating network of effective, to reduce or to eliminate the harm bringing to plant.And transcription factor plant grow and the expression regulation of defense response in have very important effect, various signal pathways that can involved in plant defense response, thereby the growing and reaction to environment etc. of regulating plant.Because an adverse circumstance associated transcription factor can regulate and control the expression of multiple downstreams functional gene, therefore utilize transcription factor to improve the resistance of trees and can obtain more desirable net effect than the transgenosis application of single downstream functional gene.

Transcription factor refers to that those are incorporated in specific manner on DNA particular sequence, can activate or suppress the protein of other genetic transcriptions.Myb transcription factor is a species specific transcription factor, when plant is under the environment stresses such as arid, low temperature and salt, myb transcription factor is induced to express, with respective element specific combination, start the expression of the functional gene that downstream is relevant to resistance, cause the accumulation of gene product, various biochemical reactions in regulating plant body, finally make plant be improved to the resistance of environment-stress.Much research shows, transcription factor has powerful function in genetic engineering, and the transcription factor of the genes encoding that overexpression is relevant can effectively improve the resistance of plant.At present, mainly concentrate in herbaceous plant about the research of myb transcription factor gene, about the research of the Cloning and Expression of the myb transcription factor gene of xylophyta less.In the research of the MYB albumen that contains single MYB structural domain, the people such as Dong find StMYB1R-1 transcription factor in potato, and the drought-induced genes involved of this gene activation, by regulating ICW disappearance to reach drought resisting object.Its research of R2R3-MYB member for most species in MYB family will be enriched much relatively, and the gene with the resistance to retrocorrelation of plant of finding at present has a lot.Only in Arabidopis thaliana, just found the many and biological genes relevant with abiotic stress, as AtMYB41 gene just belongs to R2R3-MYB class, this transcription factor has multiple regulating and controlling effect, can coerce stimulation and makes a response ABA, arid, high salt and low temperature are various.The AtMYB2 of Arabidopis thaliana and AtMYB60 are also proved the drought-enduring process of coercing of involved in plant.The AtMYB2 transcription factor of Arabidopis thaliana is expressed and is strengthened under adverse circumstance, has also strengthened the expression of arid response gene rd22 and AtADH1 simultaneously; Under drought stress, AtMYB2 also brings into play function as the transcription activator of ABA induction.AtMYB60 reacts regulating plant stomatal movement, drought stress and disease resistance thereof by dormin signal cascade.Participate in degeneration-resistant research of coercing about myb gene in monocotyledons also more.As OsMYB2 and Osmyb4 gene that from paddy rice, clone obtains.They have participated in respectively the degeneration-resistant regulation and control of difference of paddy rice.Research finds that OsMYB2 gene can be induced to express under salt, low temperature and drought condition; Osmyb4 gene in overexpression paddy rice can significantly improve the tolerance of transgenic plant to arid, high salt, UV radiation etc.In addition, the BcMYB1 gene in capsule lettuce tongue finds that through experiment this gene can strong expression under drought-induced condition, also can coerce generation replying to a certain degree to PEG, high salt, low temperature etc. simultaneously.In recent years, found the R1R2R3-MYB transcription factor with 3 repeating structure territories that plays a role in plant stress-resistance is coerced.As the R1R2R3-MYB transcription factor gene OsMYB3R-2 being cloned into from paddy rice, in Arabidopis thaliana, after this gene of overexpression, discovery transfer-gen plant is compared adjoining tree the tolerance of low temperature, arid and high salt is significantly improved.In addition, also have many myb genes to be subject to the induction of low temperature or high temperature, the frost resistance AtMYB68 gene that can strengthen transfer-gen plant as AtMYB15 gene is expressed under high temperature induction.

These results show, the by-pass cock that myb 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 multiple and degeneration-resistant relevant functional gene to express, this is to make plant stress-resistance proterties obtain a very effective approach of comprehensive improvement.And MYB class transcription factor plays an important role in stress signal transmittance process, it can regulate and control the expression of multiple functional genes relevant with plant arid, high salt and low temperature patience, this is to strengthening on the whole the resistance of plant, improve its stability, to there is huge application prospect, there is huge potential using value for the resistance of reverse of genetically engineered improvement plant.Therefore, clone the new MYB class transcription factor gene with independent intellectual property right, study its basic biological characteristics and function, to provide fundamental basis for studying whole plant stress-resistance gene regulatory network and stress response reaction mechanism, for utilizing the method improvement crop anti-adversity of plant genetic engineering, create new degeneration-resistant seeds and provide material base.

But there is no at present the relevant report about Fraxinus velutina myb transcription factor.

Summary of the invention

The present invention is directed to the deficiencies in the prior art, provide a kind of regulate and control FvMYB1 gene and the application thereof of Fraxinus velutina salt tolerance.

Regulate and control a FvMYB1 gene for Fraxinus velutina salt tolerance, nucleotide sequence is as shown in SEQ ID No.1.

By a polypeptide for above-mentioned FvMYB1 genes encoding, aminoacid sequence is as shown in SEQ ID No.2.

A kind of carrier that inserts nucleotide sequence shown in above-mentioned SEQ ID No.1.

A kind of reconstitution cell, the carrier that has inserted nucleotide sequence shown in above-mentioned SEQ ID No.1 or contained SEQ ID No.1 sequence.

Above-mentioned FvMYB1 gene, carrier or reconstitution cell are produced the application in improvement cash crop.

Beneficial effect

The present invention has cloned the FvMYB1 gene of regulation and control Fraxinus velutina salt tolerance from Fraxinus velutina, and the expression of this gene is subject to the impact of salt stress; FvMYB1 gene pairs of the present invention strengthens the resistance of plant on the whole, improve its stability, huge application prospect will be had, resistance of reverse for genetically engineered improvement plant has huge potential using value, provide fundamental basis for studying whole plant stress-resistance gene regulatory network and stress response reaction mechanism, for utilizing the method improvement crop anti-adversity of plant genetic engineering, create new degeneration-resistant seeds and provide material base.

Brief description of the drawings

Fig. 1 is the sxemiquantitative PCR electrophorogram that under condition of salt stress, Fraxinus velutina Different Organs is expressed FvMYB1 gene dosage;

Wherein: the first row electrophorogram is the sxemiquantitative PCR result of Chinese wax FvMYB1 gene; The second row electrophorogram is to contrast the pcr amplification result of carrying out using Chinese wax β-actin gene as semiquantitative internal reference;

1, root, 2, cotyledon, 3, stem, 4, leaf, 5,300mM salt stress processes the root after 24h, 6,300mM salt stress processes the cotyledon after 24h, 7,300mM salt stress processes the stem after 24h, 8,300mM salt stress processes the leaf after 24h;

Fig. 2 is that the PCR of transgene tobacco detects electrophorogram;

Wherein: M, DL2000; 1, positive control; 2, water contrast; 3-16, FvMYB1 transfer-gen plant; 17, negative control.

Embodiment

Below in conjunction with embodiment and Figure of description, technical scheme of the present invention is described further, but institute of the present invention protection domain is not limited to this.

Embodiment

1, experimental technique and step

The separation of 1.1 genes

Taking the seedling leaves of 3 weeks seedling ages of Fraxinus velutina as material, take 0.2g fresh material and grind rapidly in the mortar that is added with liquid nitrogen; Fully be ground to after finely powdered, packed in the 1.5ml centrifuge tube that diethylpyrocarbonate (DEPC) handles well, add 65 DEG C of preheated cetyl trimethylammonium bromides of 600 μ l (CTAB) extracting solution (being prepared by DEPC water), immediately violent vortex 30sec; 65 DEG C of temperature are bathed 2～5min, during this time vortex 3～5 times; After slightly cooling, add equal-volume chloroform/saturated phenol, violent vortex 60sec; 4 DEG C, the centrifugal 15min of 12000rpm; Get supernatant liquor and move in new 1.5ml centrifuge tube, add the Virahol of equal-volume precooling ,-20 DEG C of precipitation 2h; 4 DEG C, the centrifugal 15min of 12000rpm, abandons supernatant; Use successively 70% ethanol, absolute ethanol washing, after drying, add 30～50 μ l DEPC water dissolution, make RNA solution.

RNA after dissolving is carried out to purifying, and reaction system is as follows: RNA solution 20～50 μ g, 10 × Dnase I Buffer5 μ l, Dnase I(5u/ul) 1 μ l, Rnase I Inhibitor(40u/ul) 0.5 μ l, finally add DEPC H ₂o is to cumulative volume 50 μ l, in 37 DEG C of reaction 1h; Add afterwards 350 μ l DEPC H ₂o; (400 μ phenol/chloroform/primary isoamyl alcohol (volume ratio 25:24:1) l), mixes to add equivalent; Centrifugal, get upper strata water in new centrifuge tube; (400 μ chloroform l), fully mixes to add equivalent; Centrifugal, get upper strata water in new centrifuge tube; Add 1/10 volume (10 μ 3M NaAC(pH 5.2 l)); Add the cold dehydrated alcohol of 2.5 times of volumes ,-20 DEG C are spent the night; Centrifugal recovery precipitation, 70% washing with alcohol is dry; Add 20 μ l DEPCH ₂after O dissolves, electrophoresis detection.

Utilize the cDNA sequence of 3 ' RACE method amplification FvMYB1 gene.Concrete operation method is as follows:

First, mRNA reverse transcription being become to the first chain cDNA, uses the RNA LA PCRTM Kit(AMV of Takara company) test kit carries out reverse transcription, and reaction system is 10 μ l.Add successively 2 μ l MgCl ₂, 10 × RNA PCR Buffer, 1 μ l, the each 10mM of dNTP Mixture() and 1 μ l, Rnase Inhibitor 0.25 μ l, Reverse Transcriptase 0.5 μ l, OligoDt-Adaptor Primer 0.5 μ l, Total RNA (l) 4.75 μ l of 1 μ g/ μ.After system mixes, carry out reverse transcription by following condition: 30 DEG C of 10min, 50 DEG C of 30min, 99 DEG C of 5min, 5 DEG C of 10min.Carry out 3 ' RACE amplification taking the first chain cDNA as template, the primer is:

Fv-outer:5′-GTAGGGAAAGGAGATTGGAGAGGGAT-3′?SEQ?ID?NO.3

Fv-inner:5′-TTGATATTACCACTGATACGGTTTTG-3′?SEQ?ID?NO.4

In PCR reaction system, add successively 10 × PCR Buffer, 2 μ l, 2.5mM dNTP 4 μ l, cDNA 1 μ l, Auele Specific Primer 0.5 μ l(10mM), universal primer 0.5 μ l(10mM), LA Taq archaeal dna polymerase 0.1 μ l, adds ddH ₂o to 20 μ l.

PCR reaction conditions: 94 DEG C of denaturations 5 minutes; 94 DEG C of sex change 30 seconds, anneal 58 DEG C 30 seconds, extend 72 DEG C 2 minutes, 32 circulations; Extend 10 minutes eventually 4 DEG C of preservations.

PCR product, in 1wt% agarose gel electrophoresis, is cut after glue reclaims and is connected in T3 Easy vector(purchased from Beijing Quanshijin Biotechnology Co., Ltd).Linked system 5 μ l, wherein 1 μ l T3 Easy vector, 4 μ l PCR products.In 25 DEG C of connections 20 minutes, will connect product and transform DH5 α competent cell.After screening positive clone, send order-checking.Determine that according to est sequence information and Arabidopis thaliana, apple, soybean and the comparison of paddy rice amino acid in GenBank database the est sequence 5 ' end obtaining comprises FvMYB1 gene start codon ATG.3 ' the RACE sequence that order-checking is obtained and its splicing obtain the full length cDNA sequence of FvMYB1.

The expression analysis of 1.2 genes

By sxemiquantitative RT-PCR method, FvMYB1 gene is carried out to the tissue specific expression analysis under salt stress.Concrete grammar is as follows:

Taking the cDNA of the each total tissue RNA reverse transcription of Chinese wax as template, according to the special primer of document design Chinese wax β-actin sequence,

β-actin-F：5′-GCAGGGCGTGATTTAACTG-3′，SEQ?ID?NO.5

β-actin-R：5′-CTCCGATCCAGACACTGTACT-3′?SEQ?ID?NO.6

According to the FvMYB1 sequence being cloned into, design special goal gene RT-PCR reaction primer:

FvMYB1-RT-F：5′-GCTATTGTTGTTGGGTGGT-3′，SEQ?ID?NO.7

FvMYB1-RT-R：5′-GCTATTGTTGTTGGGTGGT-3′SEQ?ID?NO.8

Taking the cDNA of total RNA reverse transcription that extracts Fraxinus velutina each organ as template, carry out pcr amplification, PCR reaction conditions is: 94 DEG C of denaturation 5min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C are extended 1min, totally 25 circulations; Extend 10min in 72 DEG C.

The structure of 1.3 plant expression vectors

Carry out the checking of gene function by building pROKII-FvMYB1 carrier transformation of tobacco.Concrete operation method is as follows:

Adopt primer:

FvMYB1-S：5’-GC TCTAGACTAGCATTCGTTAATATACTTTTTA-3’?SEQ?ID?NO.9

FvMYB1-A：5’-CGC GGATCCACAGACATCAATAACATTATTCCCA-3’?SEQ?ID?NO.10

Taking the plasmid that comprises goal gene as template, the FvMYB1 gene that amplification comprises complete reading frame.

In PCR reaction system, add successively 10 × PCR Buffer, 2 μ l, 2.5mM dNTP 4 μ l, cDNA 1 μ l, Auele Specific Primer 0.5 μ l (10mM), universal primer 0.5 μ l (10mM), LA Taq archaeal dna polymerase 0.1 μ l, adds ddH ₂o to 20 μ l.PCR reaction conditions: 94 DEG C of denaturations 5 minutes; 94 DEG C of sex change 30 seconds, anneal 55 DEG C 30 seconds, extend 72 DEG C 1 minute, 32 circulations; Extend 10 minutes eventually 4 DEG C of preservations.

PCR product, in 1% agarose gel electrophoresis, is cut after glue reclaims and is connected in T3 Easy vector(purchased from Beijing Quanshijin Biotechnology Co., Ltd).Linked system 5 μ l, wherein 1 μ l T3 Easy vector, 4 μ l PCR products.In 25 DEG C of connections 10 minutes, will connect product and transform DH5 α competent cell.After PCR screening positive clone, send order-checking.

BamHI and the Xba I enzyme produced with Fermantus company, enzyme is cut the plasmid and the carrier pROKII plasmid that comprise goal gene fragment respectively, operate as follows: enzyme is cut the each 100 μ l of system, comprise 10 μ l 10 × FD Green Buffer, plasmid/carrier pROKII that 40 μ l comprise order gene, 5 μ l FD BamHI, 5 μ l FD Xba I and 40 μ l ddH ₂o.In 37 DEG C of water-bath 15min.

Enzyme is cut the sepharose DNA recovery test kit recovery gene fragment that product Great Britain, Jinan bio tech ltd produces, and operates as follows:

The sepharose that contains target DNA fragment is cut, put into 1.5ml centrifuge tube.Add the sol solutions of 3 times of volumes, melt in 50～55 DEG C of water-bath to glue.Add 500 μ l balance liquids in adsorption column, in the centrifugal 1min of 12000rpm.Mixed solution is proceeded in the adsorption column that balance is good, in the centrifugal 1min of 12000rpm, discard waste liquid in sleeve pipe.In adsorption column, add 600 μ l rinsing liquids, in the centrifugal 1min of 12000rpm, discard waste liquid.Repeat rinsing once, discard waste liquid, the centrifugal 2min of 12000rpm.To under adsorption column room temperature, dry up, then adsorption column is transferred in aseptic 1.5ml centrifuge tube.To the Elution Buffer that adds 30 μ l65 DEG C preheatings in adsorption column, the centrifugal 1min of 12000rpm reclaims DNA solution.

The gene fragment of recovery is connected in plant expression vector pROKII, operate as follows: linked system 20 μ l, comprise the pROKII carrier that 3 μ l reclaim, the gene fragment that 8 μ l reclaim, 2 μ l 10 × T4 ligase enzyme damping fluids and 1 μ l T4 ligase enzyme, finally add ddH ₂o to 20 μ l, 16 DEG C of connections of spending the night.Connect product and transform bacillus coli DH 5 alpha competent cell, after PCR screening positive clone, enzyme is cut and is identified and send order-checking.

The genetic transformation of 1.4 tobaccos

The recombinant vectors building is proceeded in Agrobacterium LBA4404 (purchased from Biovector company), operate as follows: in 200 μ l competent cells, add 1 μ l recombinant plasmid, ice bath 30min.Liquid nitrogen freezing 1min; 37 DEG C of water-bath 2～3min.Add 800 μ l YEB substratum (peptone 10g/L, yeast extract 10g/L, NaCl 5g/L, pH=7.2), 28 DEG C are shaken bacterium 3～5h, the centrifugal 5min of 6000rpm; After the resuspended precipitation of 100 μ l YEB, coat containing 100 μ g/ml kantlex, on the flat board of 50 μ g/ml Rifampins, cultivate 2～3 days for 28 DEG C.

Adopt tobacco leaf disc method to carry out the conversion of Agrobacterium.Operate as follows:

75% ethanol disinfection 1min for tobacco seed, aseptic water washing 3～5 times; With 20% clorox, to seed disinfection 10min, aseptic water washing 3～5 times, is evenly seeded on 1/2MS minimum medium, makes its germination; After seed germination (approximately two weeks), transferred in the tissue culture bottle containing 1/2MS substratum, grow after 4～5 weeks for transforming; The single colony inoculation of Agrobacterium that picking carries recombinant plasmid contains 50 μ g/ml kantlex in 5ml, in the YEP liquid nutrient medium of 50 μ g/ml Rifampins, and 28 DEG C, 200rpm incubated overnight.Be inoculated in 50ml containing in identical antibiotic fresh YEP substratum, to OD by 1:50 ₆₀₀be 0.4～0.5, after centrifugal collection thalline, equal-volume is resuspended in MS liquid nutrient medium.Get tobacco leaf, be cut into small pieces (0.5 × 0.5cm), is dipped in 5～10min in resuspended liquid by the tobacco leaf shearing, and vibration is during this time blotted surface-moisture with aseptic filter paper after leaf dish takes out, and is arranged on MS division culture medium, 28 DEG C, secretly cultivates two days.Dark cultivation two days later, selects blades arrangement on substratum at MS, and 28 DEG C, illumination cultivation 16h/d, changed a subculture every two weeks.In the time that it grows Multiple Buds, cut and put in elongation medium, make its elongation.When Multiple Buds grows to 3～5cm, transfer to root media, short its taken root.After root system development is good, move in bactericidal nurishing soil overlay film 3～5 days; Grow to and to a certain degree move in large basin, Routine Management.It is as shown in table 1 that in experiment, Tissues of Tobacco is cultivated various substratum used.

Used medium in the cultivation of table 1 Tissues of Tobacco

Note: MS substratum comprises following composition (mg/L): macroelement: NH ₄nO ₃1650, KNO ₃1900, CaCl ₂2H ₂o 440, MgSO ₄7H ₂o 370, KH ₂pO ₄700; Trace element: KI 0.83, H ₃bO ₃6.2, MnSO ₄4H ₂o 22.3, ZnSO ₄7H ₂o 8.6, Na ₂mnO ₄2H ₂o 0.25, CuSO ₄5H ₂o 0.025, CoCl ₂6H ₂o 0.025; Molysite composition: FeSO ₄7H ₂o 27.8, Na ₂eDTA2H ₂o 37.3; Organic composition: inositol 100, nicotinic acid 0.5, pyridoxine hydrochloride (vitamin B6) 0.5, vitamin (VITMAIN B1) 0.5, glycine 2.0.1/2MS medium component is except above-mentioned macroelement reduces by half, and all the other are constant.

The qualification of 1.5 transfer-gen plants and Function Identification

Taking tobacco leaf as material, the PCR that carries out transgene tobacco detects.Operate as follows:

CTAB method is extracted transgene tobacco leaves genomic DNA, and with the positive contrast of recombinant vectors, the negative contrast of wild-type tobacco genomic dna and aseptic double-distilled water, carries out PCR detection with the primer of gene specific.

In 10 μ l PCR reaction systems, add successively TaqMix 5 μ l, DNA profiling 1 μ l, the each 0.5 μ l of FvMYB1-S and FvMYB1-A primer, dd H2O 3 μ l.

PCR reaction conditions: 94 DEG C of denaturations 5 minutes; 94 DEG C of sex change 30 seconds, anneal 55 DEG C 30 seconds, extend 72 DEG C 1 minute, 32 circulations; Extend 10 minutes eventually 4 DEG C of preservations.1.0% agarose gel electrophoresis detects, and amplification obtains the positive transfer-gen plant of clone of long 933bp fragment.

2, experimental result

The clone of 2.1 FvMYB1 genes

From Fraxinus velutina, be separated to 1 MYB FvMYB1 according to homologous clone and 3 ' RACE method.This full length gene 1203bp, nucleotide sequence as shown in SEQ ID No.1,301 amino acid of encoding altogether, aminoacid sequence is as shown in SEQ ID No.2.The sequencing results demonstration, FvMYB1 protein molecular quality is 33.38kDa, iso-electric point is 9.89.N end has 1 typical MYB/SANT DNA in conjunction with territory: R1 (85-135aa).In addition, another key character of FvMYB1 sequence is to contain conservative motif SHAQKYF in the downstream of R1 structural domain.Above result shows, the albumen of FvMYB1 coding belongs to single myb transcription factor family member.

Sequence comparison in aminoacid sequence and the database of FvMYB1 gene being derived by BLASTp program, finds that with the sequence of its homology be all myb gene.FvMYB1 conserved domain and other plant, as the conserved domain similarity of potato StMYB1R-1, soybean GmMYB176, Chinese rose RhMYB, Vinca CrMYB all reaches 100%, reach respectively 98% and 94% with the similarity of apple MxMYB and rice Os MYBS3 conserved domain.And homology region all concentrates on N end R1 in conjunction with territory, and C end homology is extremely low, illustrates that separating the FvMYB1 obtaining is 1 new Chinese wax myb gene.

2.2 the expression analysis of FvMYB1 gene

Whether be subject to the impact of salt stress in order to analyze FvMYB1 genetic expression, we have detected Chinese wax in the expression amount variation tendency of FvMYB1 gene in different tissues (root, stem, cotyledon and true leaf) after 300mM NaCl processes.

Sxemiquantitative RT-PCR result shows that FvMYB1 gene all has expression in each tissue of Chinese wax, and the expression amount of gene do not have significant difference in cotyledon, stem and leaf, slightly low compared with respectively organizing at other in root.After NaCl processes 24h, compared with the control, the expression amount of FvMYB1 gene in root, cotyledon and leaf all declines to some extent, and especially in root, expression amount obviously reduces.Expression amount in stem does not have bright variation after salt is processed.Utilize Chinese wax β-actin gene to contrast the pcr amplification result of carrying out with tester accuracy as semiquantitative internal reference simultaneously.As shown in Figure 1.

The qualification of 2.3 transfer-gen plants and gene function analysis

Adopt round pcr to detect transfer-gen plant.In transfer-gen plant, amplify object fragment, in non-transgenic plant, have no object band and occur, see Fig. 2.Result shows that goal gene has been transferred in tobacco plant.

Choose increment transgenic line of the same size and wild-type plant, carry out salt stress and process 30 days.The plant height and the biomass that found that transgenic line under 100mM salt stress are higher compared with wild-type plant, and specific experiment data are as shown in table 2.

Table 2 transgene tobacco salt is processed the growth characteristics comparison after 30 days

Can be found by table 2 data, the plant height of transgene tobacco under 100mM condition of salt stress and biomass be apparently higher than wild-type, thereby the salt tolerance that the overexpression of explanation FvMYB1 gene can render transgenic plant is enhanced.

Claims (5)

1. one kind regulates and controls Fraxinus velutina salt tolerance fvMYB1gene, nucleotide sequence is as shown in SEQ ID No.1.

2. one kind by described in claim 1 fvMYB1the polypeptide of genes encoding, aminoacid sequence is as shown in SEQ ID No.2.

3. one kind is inserted the carrier of nucleotide sequence shown in SEQ ID No.1 described in claim 1.

4. a reconstitution cell, the carrier that has inserted nucleotide sequence shown in SEQ ID No.1 described in claim 1 or contained SEQ ID No.1 sequence.

5. described in claim 1 fvMYB1the application of reconstitution cell in Tobacco Salt improved properties described in carrier or claim 4 described in gene, claim 3.