CN107267526A

CN107267526A - Pseudo-ginseng myb transcription factor gene PnMYB2 and its application

Info

Publication number: CN107267526A
Application number: CN201710540061.0A
Authority: CN
Inventors: 刘迪秋; 关瑞攀; 曲媛; 葛锋; 杨晓艳; 熊吟
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2017-07-05
Filing date: 2017-07-05
Publication date: 2017-10-20
Anticipated expiration: 2037-07-05
Also published as: CN107267526B

Abstract

The invention discloses pseudo-ginseng myb transcription factor genePnMYB2And its application,PnMYB2The nucleotide sequence of gene such as SEQ ID NO：Shown in 1, myb transcription factor is encoded；The present invention is confirmed by molecular biology and functional genomics relation technological researchingPnMYB2Gene has the function of improving plant resistance to fungal disease, by anti-fungal gene of the present inventionPnMYB2It is building up on plant expression vector and is transferred to overexpression in tobacco, transgenic tobacco plant has very strong extracorporeal antifungal activity,PnMYB2Growth of the transgene tobacco of overexpression to grape seat chamber bacterium, Fusarium solani, Fusarium oxysporum, wheel branch sickle-like bacteria, ginseng rod method has obvious inhibitory action.

Description

Pseudo-ginseng myb transcription factor genePnMYB2And its application

Technical field

The present invention relates to molecular biology and genetic engineering Related Research Domain, particularly a kind of pseudo-ginseng MYB transcriptions because Subbase becausePnMYB2And its application.

Background technology

Plant can be constantly met with from extraneous various natural calamities and pest and disease damage in growth and development process, so that Cause the retarded growths such as cereal crops, industrial crops and medicinal plant, the phenomenon that the underproduction is even had no harvest.Plant disease point Disease and disease is infected for non-infect.Infect disease by biological factors such as fungi, bacterium, viruses to be caused, with infectiousness, harm Greatly, the duration is long.Phytopathogen species is various, prevents and treats especially difficult.The conventional method of disease control, which is mainly, uses agriculture Medicine and cultivate improved seeds.But be difficult solution the problem of the safety problem of people and animals and environmental pollution using during agricultural chemicals Certainly.Cultivate improved seeds the traditional breeding way used, time length, slowly effect, put into huge, Resistant germplasm and be difficult to obtain.This Both of which not can solve plant disease problem.And genetic engineering can be with quickly breeding using Protocols in Molecular Biology Disease resistance plant variety, is to strengthen the new method of plant disease-resistant ability safely, conveniently.

During natural selection and biological evolution, plant forms the molecular mechanism of distinctive controlling gene expression.Adjust Control mechanism is divided into three levels, the respectively regulation and control of transcriptional level；The regulation and control of post-transcriptional level and the regulation and control of translation skill.Transcription The factor（Transcription factor, TF）It is that a class can be combined in upstream region of gene distinguished sequence, and controlling gene is transcribed Protein.There are a variety of transcription factor protein families such as bHLH, bZIP, Zinc-finger, MYB in plant, wherein MYB makees Got most of the attention for a maximum class transcription factor.

Plant transcription factor MYB is a class regulating growth of plants, participates in cell cycle, metabolism and to biological, non- The transcription factor of biotic response activity.The 1st in plantMYBGene be 1987 from monocotyledonous plant Zea mays (Zea mays) in separation identify comeZmMYBCIGene.MYB generally existings in plant, because have in its structure one section it is conservative DNA lands, i.e. Myb are gained the name.This conserved domain is connected by 1 to 3 incomplete same R (R1, R2, R3) domains Composition.Myb transcription factor is divided into 4 classes according to the different compositions of conserved domain：1R-MYB/MYB-related；R2R3- MYB；3 R-MYB；4R-MYB（The related myb transcription factor research of the such as Xue Yingxi, Wei Jianhua, Jiang Tingbo Plant Secondary Materials growth Be in progress Agriculture of Anhui science, 2012,40 (13):7650-7655）.The conserved amino acid that R structures are included with Helix-turn- The form of spiral (Helix-Turn-Helix, HTH) participates in the transcription of gene.

Myb transcription factor participates in growth and development of plants, secondary metabolism, biology and abiotic stress defense reaction, response and planted Thing HORMONE TREATMENT（Niu Yiling, Jiang Xiuming, the progress molecules of plant transcription factors myb gene family are planted on the sunny side perhaps Thing breeding, 2016,14 (8): 2050-2059）.Will from sweet wormwood (Artemisia annua) myb geneAaMYB1Turn Enter wild sweet wormwood overexpression, as a result the artemislnin content of transgenic positive plant is significantly improved compared with wild type, it is ciliary to increase Grow and be also apparent from（Matías-Hernández L, Jiang W, Yang K et al. AaMYB1 and its orthologue AtMYB61 affect terpene metabolism and trichome development inArtemisia annua and Arabidopsis thaliana. plant journal, 2017, 90(3): 520- 534）.T-DNA is inserted into arabidopsisMYBHGene, is obtainedmybh1Mutant, the as a result aging of mutant blade goes out compared with wild type Existing delay phenomenon（Huang CK, Lo PC, Huang LF, et al. A single-repeat MYB transcription repressor, MYBH, participates in regulation of leaf senescence in Arabidopsis. plant molecular biology, 2015, 88(3): 269-286）.Will using transgenic technology From Jatropha curcas (Jatropha curcas)JcMYB2Be transferred to arabidopsis (Arabidopsis thaliana) overexpression, Salt stress is carried out to transgenosis and WT strain respectively and low temperature stress is handled, as a result the survival rate of transgenic line is above Wild type, i.e.,JcMYB2Overexpression improve the salt tolerance and lower temperature resistance of arabidopsis（Peng X, Liu H, Wang D, et al. Genome-wide identification of the Jatropha curcas MYB family and functional analysis of the abiotic stress responsive gene JcMYB2. BMC Genomics,2016,17:251）.From chrysanthemum (Chrysanthemum morifolium) in isolateCmMYB19, and use Real-time PCR detect chrysanthemum by aphid (Macroaiphoniella sanborni) take food afterCmMYB19Expression Level changes, as a result 6 h after aphid takes food, 9 h and 24 hCmMYB19Expression have difference than normal plant The up-regulation of degree（Wang Y, Sheng L, Zhang H et al.CmMYB19 Over-expression improves aphid tolerance in Chrysanthemum by promoting lignin synthesis. international journal of molecular sciences, 2017, 18(3): 619）.

Salt (salt), salicylic acid (Salicylic acid, SA) and the methyl jasmonate (Methyl of various concentrations are used respectively Jasmonate, MeJA) processing sweet cherry (Prunus avium) detect after seedlingPacMYBAExpression, qPCR result tables 1 h after bright salt stress processingPacMYBAIt is induced expression and 6h expression quantity after treatment reaches highest, is untreated control 5.5 times of group.After other SA and MeJA processingPacMYBAExpression becoming of being stepped up all is presented during whole sampling Gesture, after treatment 12 hPacMYBAExpression quantity be 5.2 times and 8.7 times of control group respectively.Data above is provedPacMYBA Respond salt, salicylic acid and methyl jasmonate Stress treatment.Will using the method for genetic engineeringPacMYBAIt is transferred to arabidopsis progress different Express in source.Handle transgenosis and wildtype Arabidopsis thaliana respectively with 250mMNaCl and find that the appearance of plant animation is substantially poor after 7 days Different, wildtype Arabidopsis thaliana blade is wilted, and growth is suppressed completely, and transgenic arabidopsis growth conditions are good, show external sourcePacMYBAImprove tolerance of the arabidopsis to salt stress.Use bacterial pathogensPseudomonas syringe pv. tomato (Pst) type strain DC3000 spore suspensions processing wild type and transgenic arabidopsis,PacMYBAOverexpression Inhibit the growth of bacterium；Trypan Blue result shows that the Disease symptoms of WT strain blade are more obvious.Normal condition The activity of peroxidase is higher than wild type in lower transgenic line, and the activity of pathogen processing rear oxidation thing enzyme is all raised, The enzymatic activity of transgenic line is still high compared with wild type.To sum up,PacMYBAOverexpression improve transgenic Arabidopsis plants pairPstDC3000 resistance（Shen X, Guo X, Guo X et al. PacMYBA, a sweet cherry R2R3-MYB transcription factor, is a positive regulator of salt stress tolerance and pathogen resistance. plant physiology and biochemistry, 2017, 112: 302-311）.

Myb gene in the present inventionPnMYB2From pseudo-ginseng (Panax notoginseng), pseudo-ginseng be Yunnan Province it is important in Medicine resource, has effects that " raw to beat ripe mend ".The Panax notoginseng Growth cycle is long, and property happiness is warm dark and damp, and disease is serious, particularly root-rot The fungal diseases such as disease, black spot, Northern leaf spot, seriously endanger pseudo-ginseng yield and the quality of medicinal material.Therefore, for the disease-resistant correlation of pseudo-ginseng The clone of gene, functional analysis and application study are particularly urgent.

The content of the invention

It is an object of the invention to provide a kind of pseudo-ginseng myb transcription factor genePnMYB2And itsPnMYB2Application, that is, exist Improve tobacco to grape seat chamber bacterium (Botryosphaeria dothidea), Fusarium solani (Fusarium solani), point Fusarium oxysporum (F. oxysporum), wheel branch sickle-like bacteria (F. verticillioides), ginseng rod method (Alternaria panaxWhetz) the application in resistance.

The present invention clones the myb transcription factor gene with antifungal activity obtained from pseudo-ginsengPnMYB2Total length base Cause,PnMYB2Nucleotide sequence such as SEQ ID NO：Shown in 1, the full length gene is 1209bp, includes 864bp opening 5 ' the non-translational regions (untranslated region, UTR) and 194bp 3 ' UTR of reading frame, 151bp, coding such as SEQ ID NO：The protein of amino acid sequence shown in 2.

Myb transcription factor gene of the present inventionPnMYB2Code area be sequence table SEQ ID NO：152- in 1 Nucleotide sequence shown in 1015.

The global cDNA fragment of an antimycotic related gene for present invention separation clone pseudo-ginseng, passes through Agrobacterium tumefaciems (Agrobacterium tumefaciens) target gene is transferred to overexpression in recipient plant by mediation, and by further Whether the experimental verification gene has antimycotic activity, is that the later-stage utilization improvement of genes tobacco and other plant resist fungi The ability of disease lays the foundation；This unnamed gene is by inventorPnMYB2。

Included the present invention relates to separationPnMYB2DNA fragmentation and identify its function, wherein the DNA fragmentation such as sequence table It is shown,PnMYB2Full-length cDNA is 1209bp, 5 ' non-translational regions of ORFs, 151bp comprising a 864bp (untranslated region, UTR) and 194bp 3 ' UTR, wherein ORF encode an albumen with 287 amino acid Matter.BLASTn analysis results are shownPnMYB2Partial sequence and lead a cow (Ipomoea nil) transcription repression gene (XM_ 019328930) have 86% similitude, with tea tree (Camellia sinensis) transcription factor protein gene (HQ660373.1) have 84% similitude, with alpine ash (Eucalyptus grandis) transcription repression gene (XM_ 010063679) there is 82% similitude.Protein homology analysis showsPnMYB2The protein sequence of coding and cucumber (Daucus carota), the transcription factor protein of tea tree have respectively 69% and 64% similitude.PnMYB2The protein of coding Sequence has the conserved domain of MYB superfamilies, and this shows its MYB albumen belonged in pseudo-ginseng.Overexpress sequence table SEQ ID NO：Sequence shown in 1 can strengthen tobacco to grape seat chamber bacterium, Fusarium solani, Fusarium oxysporum, wheel branch sickle-like bacteria, ginseng chain The resistance of lattice spore.

It is above-mentionedPnMYB2Gene can apply to improve the antifungal property of tobacco, and concrete operations are as follows：

(1) using amplificationPnMYB2Special primer, from inoculation Fusarium solani after 12 h Roots of Panax Notoginseng in extract total serum IgE, lead to Cross reverse transcriptase chain reaction (reverse transcription-polymerase chain reaction, RT- PCR) amplifyPnMYB2Full length coding region, be subsequently attached on pGEM-T carriers, through sequencing obtain have purpose base The clone of cause；

(2) restriction enzyme is usedEcoRI andBamHI digestions pGEM-T-PnMYB2Carrier and plant expression vector PCAMBIA2300S, target gene fragment and carrier large fragment are obtained by glue reclaim；It will be obtained againPnMYB2Genetic fragment It is connected with pCAMBIA2300S carrier segments, builds plant overexpression vector；Constructed recombinant vector is passed through into crown gall afterwards Agriculture bacillus mediated be transferred in tobacco is expressed；

(3) there is kalamycin resistance gene on recombinant vector T-DNA, screened and converted with the differential medium of addition kanamycins Son, and real transfer-gen plant is obtained by PCR and RT-PCR detections, analysis transfer-gen plant is for plant pathogenic fungi Resistance, finally filter out the transfer-gen plant being remarkably reinforced to fungus resistant.

The present invention provides a kind of new method to improve the resistance of plant against fungal disease, is trained by genetic engineering means Traditional breeding method can be overcome the shortcomings of by educating disease-resistant plants, and not only breeding cycle shortens, and simple to operate, is readily available Gao Kangcai Material；From pseudo-ginseng in the present inventionPnMYB2Gene can strengthen resistance of the plant to several disease funguses, by the channel genes cigarette In grass, new varieties and new material with fungus resistant can be produced.Using technique for gene engineering cultivate resistance plant kind and Material has obvious advantage and the importance do not replaced.It can be not only large-scale production crop, flowers, medicinal plant Deng providing convenient, the use of chemical pesticide is reduced, can also be that agricultural production is cost-effective, reduce environmental pollution, therefore this hair It is bright that there is wide market application foreground.

Brief description of the drawings

Fig. 1 is part in the present inventionPnMYB2The PCR testing results of transgene tobacco genomic DNA, wherein Marker： DL2000 DNA Marker (Dalian is precious biological), by 2,000bp, 1,000bp, 750bp, 500bp, 250bp and 100bp six Bar DNA fragmentation is constituted；Positive control：Plasmid pGEM-T-PnMYB2Reacted for the PCR of template；WT：Non-transgenic tobacco is (wild Type) STb gene be template carry out PCR；

Fig. 2 is some positive in the present inventionPnMYB2In transgene tobaccoPnMYB2The expression analysis result figure of transcriptional level, its Middle Marker：DL2000 DNA Marker (Dalian is precious biological)；WT：Non-transgenic tobacco total serum IgE reverse transcription cDNA is template PCR primer；Positive control：Plasmid pGEM-T-PnMYB2For the PCR primer of template；

During Fig. 3 is the present inventionPnMYB2The fungistatic effect figure of transgene tobacco extracorporeal antifungal activity；Wherein a, b, c, d, e are illustrated In fungi be Fusarium oxysporum, wheel branch sickle-like bacteria, grape seat chamber bacterium, Fusarium solani, ginseng rod method respectively；WT is wild The total protein of type tobacco；Buffer is blank control, i.e., without protein control (being used for the buffer solution for extracting albumen).

Embodiment

Below by drawings and examples, the present invention is further described, but the scope of the present invention is not limited in described Hold, method operating according to a conventional method unless otherwise specified in the present embodiment, the use of agents useful for same unless otherwise specified is normal Rule reagent or the reagent configured according to a conventional method.

Embodiment 1：PnMYB2Full length cDNA clone and sequence analysis

The root of pseudo-ginseng is inoculated with Fusarium solani, total serum IgE is extracted with the root of 12 h after inoculation, with liquid nitrogen by treated pseudo-ginseng Root grind into powder, is then transferred in centrifuge tube, and total serum IgE is extracted using guanidine isothiocyanate method；Using M-MLV reverse transcriptases (promega) using total serum IgE as the chains of templated synthesis cDNA first, reaction system and operating process are：5 μ g total serum IgEs are taken, are sequentially added 50 ngoligo (dT), 2 μ LdNTP Mix (2.5mM each), with DEPC water by reaction volume polishing to 14.5 μ L；Mix Afterwards, it is rapid in the min of cooled on ice 5 after 70 DEG C of min of heat denatured 5, then sequentially add 45 × First-stand of μ L Buffer, 0.5 μ L RNasin (200U), 1 μ L M-MLV (200U), are mixed and brief centrifugation, 42 DEG C of h of warm bath 1.5, are taken out 70 DEG C are heated 10 min, terminating reaction afterwards；- 20 DEG C are placed in after the synthesis of the chains of cDNA first to save backup.

The first chain cDNA using synthesis is template, amplifying target genesPnMYB2, upstream and downstream primer sequence used is respectively 5 ' GAGCTAGAAGAAATTGACGACGATG3 ' and 5 ' GAGTACAGTTCTATTCATTTTCCCAAC3 '.Using Advantage^TM 2 PCR Enzyme (Clontech) amplify target gene.PCR reaction conditions：95℃ 1min；94 DEG C of 30s, 58 DEG C 30s, 72 DEG C of 1min, 32 circulations；72℃ 5min.Reaction system (20 μ L) is 1 μ L cDNA, 2 10 × Advantage of μ L 2 PCR Buffer, 1.8 μ L dNTP Mix (10mM each), 0.2 μ L forward primers (10 μM), 0.2 μ L reverse primers (10 μ M)、0.2μL Advantage 2 PCR Polymerase Mix、14.6μL PCR-Grade water.After PCR terminates, 8 μ are taken L enters row agarose gel electrophoresis, specificity and size to detect amplified production.

Resulting PCR primer only has a DNA band, directly carries out TA clones to PCR primer, and the kit used is PGEM-T vector kit (Promega), reaction system and operating process are：1.5 μ L PCR primers are taken, 1 μ L are sequentially added PGEM-T vector (50 ng/ μ L) and 2.5 μ L 2 × Ligation solution I, are placed in 16 DEG C overnight instead after mixing Should.Connection product is transferred in bacillus coli DH 5 alpha competence by heat-shock transformed method.With containing ampicillin The LB solid medium screening positive clones of (ampicillin, Amp).Several single bacterium colonies are selected, are shaken after bacterium with amplificationPnMYB2Special primer detection multiple cloning sites insertionPnMYB2Clone.Obtained positive colony is sequenced, finally ObtainPnMYB2Full-length cDNA is 1209 bp, passes through NCBI ORF finder (http:// Www.ncbi.nlm.nih.gov/gorf/gorf.html) analysis finds its opening code-reading frame comprising a 864bp (see sequence List).PnMYB2One protein PnMYB2 containing 287 amino acid of coding, its molecular weight is about 32.6 KDa, and isoelectric point is 9.16.Analyzed by bioinformatics software SignalP 4.1PnMYB2The protein sequence of coding, detects whether it has N-terminal Signal peptide.As a result being shown in PnMYB2 does not have signal peptide, therefore speculates that PnMYB2 albumen is not secretory protein.

Embodiment 2：Plant overexpression vector is built

Extracted and inserted using a small amount of extraction agent boxes of SanPrep pillar DNAs (Shanghai life work)PnMYB2Escherichia coli matter Grain pGEM-T-PnMYB2And plant expression vector pCAMBIA2300S plasmids, take 1 μ L to be used to agarose gel electrophoresis examine Survey the integrality and concentration level for extracting plasmid.Use restriction enzymeBamHI andEcoRI is respectively to plasmid pGEM-T-PnMYB2Double digestion (100 μ L systems) is carried out with pCAMBIA2300S, reaction system and operating process are：20 μ L are taken respectively pGEM-T-PnMYB2With pCAMBIA2300S plasmids, sequentially add 10 μ 10 × H of L buffer, 5 μ LEcoRI、5 μLBamHI、60 μL ddH₂O, is centrifuged in short-term after mixing, is placed in 37 DEG C of reaction overnights.All digestion products progress agarose is coagulated Gel electrophoresis, then using kit pairPnMYB2Fragment and pCAMBIA2300S carriers large fragment carry out glue reclaim respectively, take 1 μ L recovery products detect the size and concentration for reclaiming fragment by agarose gel electrophoresis, are placed in -20 DEG C and save backup.

Using T4 DNA Ligase (TaKaRa), by recoveryPnMYB2 DNA fragmentation and pCAMBIA2300S carrier-pellets Section is connected, and reaction system (20 μ L) and operating process are：Take 10 μ LPnMYB2 DNA fragmentation sequentially adds 2 μ L PCAMBIA2300S carrier DNAs, 2 μ L 10 × T4 DNA Ligase Buffer, 1 μ L T4 DNA Ligase, 5 μ L ddH₂O, is centrifuged in short-term after mixing, then 16 DEG C of water-bath reaction overnights.Then connection product is transferred to greatly using heat-shock transformed method In enterobacteria DH5 α, with the solid medium screening positive clone containing 50 mg/L kanamycins (kanamycin, Km).Select Single bacterium colony shakes bacterium, is expanded by template of bacterium solutionPnMYB2Special primer enter performing PCR, pick outPnMYB2With The clone that pCAMBIA2300S is successfully connected, in obtained positive strain adding glycerine is placed in -80 DEG C and saves backup.

Extracted using kit and purify the pCAMBIA2300S- in above-mentioned bacillus coli DH 5 alphaPnMYB2Plasmid.Then With frozen-thawed method by the plant expression vector pCAMBIA2300S- of above-mentioned structurePnMYB2It is transferred to prepared Agrobacterium tumefaciems In LBA4404 competent cells.Operating procedure is：Take 0.2 μ g pCAMBIA2300S-PnMYB2Plasmid, which is added, contains 200 μ L In the centrifuge tube of competent cell, the min of ice bath 5 after gently mixing is then continued in liquid nitrogen and is freezed 1 min, is then immediately placed in 37 DEG C of min of water-bath 5, then the min of ice bath 2, add 500 μ L LB Liquid Cultures and are based on 28 DEG C of h of shaken cultivation 4 afterwards.Will activation Agrobacterium afterwards is applied on the LB solid mediums containing 50 mg/L Km, and 28 DEG C are inverted culture.Picking individual colonies shake bacterium, then use AmplificationPnMYB2Specific primer enter performing PCR reaction, detect pCAMBIA2300S-PnMYB2Whether it is transferred in Agrobacterium.It is right Saved backup in being placed in -80 DEG C after positive colony, addition glycerine.

Embodiment 3：Agriculture bacillus mediated Genetic Transformation in Higher Plants and genetically modified plants screening

The transgene receptor of this experiment be tobacco (Nicotiana tabacum).By tobacco seed with 75% alcohol-pickled 30 After s, sterile water washing with 0.1% HgCl₂Soak 8 min, then again with sterile water washing several times, be seeded in 1/2 MS training Support on base, 28 DEG C of light culture 5-8 d go to illumination box (25 DEG C, 16h/d illumination), monthly cultivated later with MS after germination Base subculture is once.

That preservation is taken out from -80 DEG C of refrigerators contains pCAMBIA2300S-PnMYB2The Agrobacterium LBA4404 bacterium of plasmid Kind, take 20 μ L to be inoculated in the LB fluid nutrient mediums that 5 mL contain 50 mg/L Km and 20 mg/L rifampins, 28 DEG C of cultures are extremely Culture medium is muddy.The muddy bacterium solutions of 1 mL are drawn to the LB solid mediums containing 50 mg/L Km, 28 DEG C of 48 h of culture.With The Agrobacterium on LB solid mediums is scraped into the appropriate MGL liquid for being inoculated in the acetosyringone for being attached with 20 mg/L afterwards to train Support in base, 28 DEG C of shaken cultivation 5-8 h are to activate Agrobacterium.

Tobacco aseptic seedling young tender leaf is taken to be cut into about 1 cm²Leaf dish, be completely soaked in it is above-mentioned containing activation Agrobacterium MGL In fluid nutrient medium, 25 DEG C of 15 min of dip-dye.The bacterium solution on leaf dish surface is blotted with aseptic filter paper, leaf dish is placed in co-cultivation base On, co-cultured 2 days under 22 DEG C of no light conditions.The co-cultivation base of Transformation of tobacco is MS+0.02 mg/L 6-BA+2.1 mg/L NAA The g/L agar of+30 g/L sucrose+6.

Leaf dish after co-cultivation is gone into seedling differentiation in the MS screening and culturing mediums added with antibiotic, while screening transgenic Plant.Tobacco screening and culturing medium is the g/L agar+50 of MS+0.5 mg/L 6-BA+0.1 mg/L NAA+30 g/L sucrose+6 Mg/L Km+200 mg/L cephalosporins (cefotaxime sodium salt, Cef)；Blake bottle is shifted during screening and culturing To illumination box culture (25 DEG C, 16h/d illumination, 8h/d is dark).Contain 50 mg/L Km and 200 after being used after the budding of tobacco length Mg/L Cef MS culture mediums (the g/L agar+of MS+30 g/L sucrose+6) squamous subculture.

The genomic DNA of transgenic tobacco plant blade is extracted using CTAB methods, takes genomic DNA obtained by 1 μ L to carry out fine jade Its integrality of sepharose electrophoresis detection and concentration.Used by template of the genomic DNA of transfer-gen plantPnMYB2Special draw Thing enters performing PCR reaction.After PCR terminates, 8 μ L products are taken to be used to agarose gel electrophoresis detect positive transgenic plant.Part The amplification of Transgenic Tobacco plant as shown in figure 1,PnMYB2Transgene tobacco screens 51 plants of positive transgenic plant altogether.

Embodiment 4：In transgene tobaccoPnMYB2Expression analysis and transfer-gen plant antifungal activity analysis

The tender leaf of positive transgenic plant and non-transgenic tobacco (wild type) is taken to extract total serum IgE, reverse transcription generation respectively The chains of cDNA first, and expanded as templatePnMYB2Special primer enter performing PCR, according to each transgenosis of PCR interpretations of result In plantPnMYB2The expression quantity of transcriptional level.Total RNAs extraction and RT-PCR method are in the same manner as in Example 1.PCR terminates Afterwards, 8 μ L are taken to be used for agarose gel electrophoresis, the testing result of part individual plant as shown in Fig. 2 detect 38 transgenosis altogether In individual plantPnMYB2In transcriptional level great expression, the numbering of these individual plants is 1~38.

Several fungies that laboratory is preserved are inoculated in PDA solid mediums (200 g/L potatos, 15 g/L agar, 20 G/L glucose) on, 28 DEG C of light cultures add albumen when colony growth to diameter is about 2 ~ 3cm, analyze transfer-gen plant body Outer antifungal activity.In order to prevent albumen that other living contaminantses are extracted, whole vegetable protein extraction process is sterile behaviour Make.Take 1 g transgene tobaccos individual plant (numbering is respectively 5,9,16,27) and wild-type leaves to be put into mortar first, add 1 ML protein extracts (1M NaCl, 0.1M sodium acetates, 1% PVP, pH6.0), are fully ground.It is transferred in 1.5 mL centrifuge tubes, mixes 4 DEG C stand overnight after even.4 DEG C of 30 min of centrifugation (12,000 g/min), take supernatant in 1.5 new mL centrifuge tubes, and take It is appropriate to determine total protein concentration with UV detector.The total protein concentration of transgenosis and WT lines is adjusted to 0.2 μ g/ μ L, then take 20 μ L drops on the aseptic filter paper of each fungi culture medium respectively.Except addition on the flat board of each fungi The total protein of different transgenic tobacco plants, while the total protein and blank control (protein extraction of parallel addition wild-type tobacco Liquid).The situation of 28 DEG C of cultures observation fungi growth after a few days, and evaluate accordinglyPnMYB2Transgene tobacco it is external antimycotic Activity, as a result as shown in figure 3,PnMYB2Transgene tobacco albumen is to grape seat chamber bacterium, Fusarium solani, Fusarium oxysporum, wheel Branch sickle-like bacteria, the growth of ginseng rod method have obvious inhibitory action.

Sequence table

<110>Kunming University of Science and Technology

<120>Pseudo-ginseng myb transcription factor gene PnMYB2 and its application

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Lys Arg Lys Leu Ile Ser Arg Gly Leu Asp Pro Gln Thr His Arg Pro

115 120 125

Leu Asn Ala Thr Ala Thr Ala Ala Thr Ala Ile Thr Ala Thr Ser Leu

130 135 140

Asp Phe Arg Asn Thr Val Pro Ser Asn Ile Ile Pro Thr Glu Asn Asn

145 150 155 160

Ile Tyr Lys Leu Lys Thr Glu Ser Leu Glu Asp Gly Asn Cys Ser Ser

165 170 175

Ser Thr Thr Glu Glu Thr Gln Gln His Gln Gln Gln Gln His Gln Gln

180 185 190

Gln Tyr Phe Ala Lys Phe Gln Asn Ser Gln Val Leu Asp Leu Glu Leu

195 200 205

Ser Ile Gly Leu Pro Thr Ser Arg Thr Gln Thr Asn Asp Ser Ser Leu

210 215 220

Ser Val Asn Ser Ile Glu Ser Asn Val Arg Arg Glu Phe Met Met Val

225 230 235 240

Ala Pro Pro Leu Pro Val Leu Ser Thr Thr Val Ala Pro Arg Met Cys

245 250 255

Leu Cys Trp Lys Leu Gly Phe Gln Lys Gly Gly Gln Gln Gln Gln Gln

260 265 270

Leu Cys Ser Asn Cys Lys Ser Thr Ser Gly Phe Tyr Arg Cys Cys

275 280 285

<210> 3

<211> 25

<212> DNA

<213>Artificial sequence

<400> 3

gagctagaag aaattgacga cgatg 25

<210> 4

<211> 27

<212> DNA

<213>Artificial sequence

<400> 4

gagtacagtt ctattcattt tcccaac 27

Claims

1. a kind of pseudo-ginseng myb transcription factor genePnMYB2, it is characterised in that：Its nucleotide sequence such as SEQ ID NO：Shown in 1.

2. the pseudo-ginseng myb transcription factor gene described in claim 1PnMYB2Tobacco is being improved to grape seat chamber bacterium (Botryosphaeria dothidea), Fusarium solani (Fusarium solani), Fusarium oxysporum (F. oxysporum), wheel branch sickle-like bacteria (F. verticillioides), ginseng rod method (Alternaria panax whetz) Application in resistance.