CN103194457B - Lilium regale germin-like protein gene LrGLP2 and application thereof - Google Patents

Lilium regale germin-like protein gene LrGLP2 and application thereof Download PDF

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CN103194457B
CN103194457B CN201310143928.0A CN201310143928A CN103194457B CN 103194457 B CN103194457 B CN 103194457B CN 201310143928 A CN201310143928 A CN 201310143928A CN 103194457 B CN103194457 B CN 103194457B
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lrglp2
plant
gene
protein
sprouted
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刘迪秋
李红丽
何华
张南南
葛锋
陈朝银
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Kunming University of Science and Technology
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Abstract

The invention discloses a lilium regale germin-like protein gene LrGLP2 with antifungal activity. The gene LrGLP2 has the nucleotide sequence as shown in SEQ ID:1 and encodes germin-like protein. A relevant technology of functional genomics proves that the gene LrGLP2 has a function of improving the plant antifungal activity. The antifungal gene LrGLP2 is constructed to a plant expression vector and is transferred into tobacco to perform overexpression; and the transgenic tobacco plant has strong in vitro antifungal activity. The LrGLP2 transgenic tobacco protein has different degrees of inhibition effects on the growth of botryosphaeria dothidea, phomopsis fungi, fusarium oxysporum and botrytis cinerea.

Description

Lilium regale wilson class is sprouted fibroin gene LrGLP2 and application thereof
Technical field
The class with anti-mycotic activity that the present invention relates to molecular biology and gene engineering technology field, particularly lilium regale wilson is sprouted fibroin gene lrGLP2and application.
Background technology
Fungal disease is a class maximum in Plant diseases, accounts for 70～80% of Plant diseases, and much harm is heavy, widespread Plant diseases is all fungus-caused, and causes the massive losses of crop yield.The ordinary method of controlling fungal diseases of plants mainly contains 3 kinds: the one, use chemical bactericide, and the 2nd, seed selection resistance new variety, the 3rd, adopt rational cropping system, avoid infecting soil and the propagation with cause of disease vegetable material etc.Although these methods have obtained certain effect, all can not fundamentally solve fungal disease problem.Along with generation and the development of recombinant DNA technology, transgenic technology has been opened up the new way of plant breeding.In recent years, cultivate plant variety or the material of resistance to fungal disease by gene engineering method and obtained first-stage success, be expected fundamentally to solve fungal disease problem.
Pathogenesis-related proteins (pathogenesis-related protein, PR) is ubiquitous class Buchner's bodies in higher plant, and the stimulation of pathogenic agent or other external factors can induce it to express, and in plant defense, plays a significant role.As a member of pathogenesis-related proteins family, class sprouts fibroin (germin-like protein, GLPs) and plant is resisted allogenic material injury and multiple environment stress is closely related.GLPs be a ubiquitous class and wheat in plant ( triticum aestivum) sprout the soluble glycoprotein that element (germin) sequence height is similar, be positioned at extracellular matrix, most GLPs are stable oligopolymer (Druka A, Kudrna D, Kannangara CG, von Wettstein D, Kleinhofs A, Physical and genetic mapping of barley( hordeum vulgare) germin-like cDNAs. Proc Natl Acad Sci USA, 2002,99:850 ~ 855).GLPs and germin structural similitude, all contain beta sheet barrel-like structure territory, therefore thinks that the two all belongs to cupin superfamily.GLPs has spatial and temporal expression characteristic and different functions in different species, mainly participates in multiple physiological and biochemical procedure with the form of enzyme, acceptor and structural protein.Wherein, enzyme comprises oxalate oxidase (oxalate oxidase, OXO), superoxide-dismutase (superoxide dismutase, SOD), ADP glucose Pyrophosphate phosphohydrolase/phosphodiesterase (ADP glucose pyrophosphatase/phosphodiesterase, AGPPase) etc., acceptor is as androgen binding protein (androgen binding protein, ABP19/20) hormone receptor, Rhicadhesins acceptor etc.At present the GLPs of different plants is divided into 3 subclass: the 1st subclass is the true element (true germin) of sprouting, mainly comprises the sprouting element of wheat, barley and the GLPs of some other cereal grasss, they have the double activity of SOD and OXO; The GLPs of the 2nd subclass is mainly other Cereals, gymnosperm and the plant of Solanaceae etc. that come from except Wheat and barley, and the main and plant resistance to oxidation of this subclass has been coerced direct relation; The GLPs of the 3rd subclass mainly comprises that some relevant to growth hormone metabolism regulate albumen, their (Khuri Ss relevant to flower inducing function the physiological rhythm of plant, Bakker FT, Dunwell JM, Phylogeny, function, and evolution of the cupins, a structurally conserved, functionally diverse superfamily of proteins. Mol Biol Evol, 2001,18:593 ~ 605).
Majority concentrates on the expression of GLPs in apoplast to the research of GLPs defense mechanism, and by the effect of cell walls, pathogenic agent is made to early response.Be subject to after fungi infestation, the up-regulated of GLPs in vegetable cell, and pass through H 2o 2signal pathway carrys out the defence of regulating plant to fungi.Quantitative genetics research shows that GLPs family member has the disease resistance of wide spectrum.On No. 8 karyomit(e)s of paddy rice, find that there is 12 close linkages GLPs together, interfere and make coding by RNA osGLP8-6, osGLP8-7, OsGLP8-11gene silencing after, paddy rice to Pyricularia oryzae ( magnaporthe oryzae) and sheath blight fungus ( rhizoctania solani) infect and all become more responsive (Manosalva PM, Davidson RM, Liu B, Zhu X, Hulbert SH, Leung H, Leach JE, A germin-like protein gene family functions as a complex quantitative trait locus conferring broad-spectrum disease resistance in rice. Plant Physiol, 2009,149:286 ~ 296).GLPs gene in barley hvGER4dor hvGER5atransient overexpression, or interfere and make by RNA hvGER3aor hvGER5athe instantaneous silence of gene, can protect barley epidermic cell to avoid pathogenic fungi blumeria graminisf. sp. hordeiinvasion and attack, hvGER4dgene silencing cause allergy, and hvGER4dwith hvGER5aall new extracellular SOD (Zimmermann G, B umlein H, Mock HP, Himmelbach A, Schweizer P, The multigene family encoding germin-like proteins of barley. Regulation and function in basal host resistance. Plant Physiol, 2006,142:181 ~ 192).Grape ( vitis vinifera) GLPs gene vvGLP3can be by pathogenic fungi erysiphe necatorat infection site specificity abduction delivering; Meanwhile, Subcellular Localization shows, vvGLP3be positioned on cell walls with the recombinant protein VvGLP3:GFP of green fluorescent protein (green fluorescent protein, GFP), by with histidine-tagged vvGLP3proceed to Arabidopis thaliana overexpression, therefrom separate VvGLP3 recombinant protein have SOD activity (Godfrey D, Able AJ, Dry IB, Induction of a grapevine germin-like protein ( vvGLP3) gene is closely linked to the site of erysiphe necator infection:a possible role in defense Mol Plant Microbe Interact, 2007,20:1112 ~ 1125).
Overexpression beet ( beta vulgaris) GLPs gene bvGLP-1arabidopis thaliana, plant produce H 2o 2increase, and to two kinds of pathogenic fungies verticillium longisporumwith r. solaniresistance significantly strengthen, but do not affect seedling and useful endogenetic fungus piriformospora indicainteraction; Meanwhile, bvGLP-1overexpression activate a series of defence associated protein as the transcriptional level of PR-1, PR-2, PR-3, PR-4 and PDF1.2 (Knecht K, Seyffarth M, Desel C, Thurau T, Sherameti I, Lou B, Oelm ü ller R, Cai D, Expression of bvGLP-1encoding a germin-like protein from sugar beet in aarabidopsis thalianaleads to resistance against phytopathogenic fungi. Mol Plant Microbe Interact, 2010,23:446 ~ 457).When wheat is subject to erysiphe graminisf. sp. triticiwhile infecting, therefrom clone obtains a GLPs gene fragment taGLP5(FJ594470), this gene is positioned on the 5A karyomit(e) of wheat, quantitative fluorescent PCR analytical results shows that the expression of this gene is induced by powdery mildew, and connect after bacterium before 24 h the expression amount in disease-resistant plant higher than the expression amount in disease plant, imply that this gene participates in the defensive raction (Wang Junmei of wheat to powdery mildew, Sun Yanfei, Liu Hongyan, Kang Zhensheng, Xu Hongming, the wheat class of powdery mildew induction is sprouted clone, location and the expression analysis of fibroin. Scientia Agricultura Sinica, 2009,42:3104 ~ 3111).In addition paddy rice, osGLP1downward express and cause that plant is obviously downgraded and the change of cellular form, cause the generation of the fungal disease such as banded sclerotial blight and rice blast significantly to increase simultaneously; And incite somebody to action osGLP1proceed in tobacco and express, with different concns fusarium solanibacterium liquid is processed transgene tobacco, and less visible damage does not appear or only occur in result demonstration transgene tobacco blade, and osGLP1anti-oxidant defense response and H that relevant SOD activity mediates 2o 2the cell wall constituent such as a large amount of accumulation and xylogen between relevant (the Banerjee J of crosslinked action, Maiti MK, Functional role of rice germin-like protein1 in regulation of plant height and disease resistance. Biochem Bioph Res Commun, 2010,394:178 ~ 183).
Class of the present invention sprout fibroin gene from lilium regale wilson ( lilium regalewilson).Lilium regale wilson has another name called regallity, is China endemic species, per nnial herb, and the river valley that is only distributed in Sichuan western Minjiang River Basin height above sea level 800～2700m, in the rock seam on hill-side, has the extremely strong characteristic such as antimycotic, antiviral.
Summary of the invention
The object of this invention is to provide a kind of full-length gene that obtains the class sprouting fibroin with anti-mycotic activity of cloning from lilium regale wilson lrGLP2, class is sprouted fibroin gene lrGLP2nucleotide sequence as shown in SEQ ID:1, this full length gene is 921bp, has open reading frame (ORF), the 5 ' non-translational region of 49bp and the 3 ' non-translational region of 218bp of 654bp, coding protein of aminoacid sequence as shown in SEQ ID:2.
Class described in the present invention is sprouted fibroin gene lrGLP2coding region be other DNA sequence dnas of aminoacid sequence protein shown in the nucleotide sequence shown in 50-703 position or coding SEQ ID NO:2 in sequence table SEQ ID:1.
The global cDNA fragment of an antimycotic genes involved of separating clone lilium regale wilson of the present invention, utilize agrobacterium tumefaciens ( agrobacterium tumefaciens) mediated method proceeds to goal gene in recipient plant and overexpression, verify by further experiment whether this gene has antimycotic activity, the ability of resisting fungal disease for this improvement of genes tobacco of later-stage utilization and other plant lays the foundation, and contriver by this unnamed gene is lrGLP2.
GLPs is the outer glycoprotein of the class born of the same parents in PRs family, in plant disease-resistant defense response, play an important role, GLPs mainly works by OXO, SOD, AGPPase, SOD and OXO are that ROS important in plant materials removes enzyme, in plant opposing oxidative stress process, play an important role, SOD changes hydrogen peroxide and oxygen molecule into super-oxide, and the reaction of its mediation is one of Antioxidative Defense System main in plant, and peroxidase changes water into hydrogen peroxide subsequently; OXO produces CO by catalysis oxalic acid 2and H 2o 2, the H that SOD and OXO catalysis produce 2o 2can strengthen by cellulose crosslinked effect the structure of cell walls, and the oxidation cross-linked formation mastoid process of catalysis cell walls, delay and stop intrusion and the diffusion of pathogenic bacteria, avoid subinfection again with Cell protection.In addition H, 2o 2can optionally participate in signal cascade reacts to cause the defense response of plant or directly forms a kind of antibacterial effect.In activation Defense gene expression process, H 2o 2can also cause allergy as a kind of second messenger, and infected position is reduced external microbe by allergy generating routine sexual cell apoptosis and is invaded the loss bringing.
The present invention relates to separate and comprise lrGLP2dNA fragmentation and identify its function, the plant with this gene fragment has the phenotype of the specific fungal infection of opposing to a certain extent, wherein said DNA fragmentation, as shown in sequence table SEQ ID:1, carries out sequential analysis to this gene, shows lrGLP2full length cDNA sequence is 921bp, its open reading frame that comprises a 654bp (Open reading frame, ORF), the 5 ' non-translational region (untranslated region, UTR) of 49bp and the 3 ' UTR of 218bp, one of ORF coding has 217 amino acid whose protein.LrGLP2 proteins encoded has class and sprouts the conserved domain of fibroin, with from wheat ( triticum aestivum), small liwan moss ( physcomitrella patens), pea ( pisum sativum), Sunflower Receptacle ( helianthus annuus) and the class of other species to sprout fibroin height similar, show that its class belonging in lilium regale wilson sprouts element.Sequence shown in overexpression sequence table SEQ ID can significantly strengthen the resistance of tobacco to grape seat chamber bacterium, Phomopsis fungi, Botrytis cinerea, Fusarium oxysporum.
Another object of the present invention is by lilium regale wilson glutathione S-transferase gene lrGLP2be applied in and improve tobacco in the resistance of grape seat chamber bacterium, Phomopsis fungi, Fusarium oxysporum, Botrytis cinerea, concrete operations are as follows:
(1) adopt amplification lrGLP2special primer, in the lilium regale wilson root from inoculation Fusarium oxysporum, extract total RNA, amplify by RT-PCR lrGLP2full length coding region, be then connected on pMD-18T carrier, obtain and there is the clone of goal gene through order-checking;
(2) use restriction enzyme bamhI and ecorI enzyme is cut pMD-18T- lrGLP2carrier, is reclaimed and is obtained goal gene fragment by glue.Plant expression vector pCAMBIA2300s is processed with same restriction endonuclease, glue reclaims and obtains required carrier large fragment, gene fragment and plant expression vector pCAMBIA2300s that glue is reclaimed) carrier large fragment is connected, build plant overexpression vector, then constructed recombinant vectors is expressed by Agrobacterium tumefaciens mediated proceeding in tobacco;
(3) the resistance marker screening transformant to have on recombinant vectors T-DNA, and by polymerase chain reaction (Polymerase Chain Reaction, PCR) and RT-PCR(Reverse Transcription-PCR) obtain real transfer-gen plant, analyze the restraining effect of transgenic plant albumen to fungal growth, finally filter out the transfer-gen plant that fungus resistant is obviously strengthened.
The present invention provides a kind of new method for improving plant to the resistance of fungal disease, is cultivated disease-resistant plants and can be overcome the deficiency of traditional breeding method by genetic engineering means, not only breeding cycle shortening, and also simple to operate, easily obtain high resistance material.The present invention is from lilium regale wilson lrGLP2gene can strengthen the resistance of plant to fungi, and this gene is imported in tobacco, can produce new variety and the novel material with fungus resistant.Utilize genetic engineering technique to reduce the disease that fungi brings and there is obvious advantage and irreplaceable importance.It can be provided convenience for scale operation crop, flowers etc., reduces in a large number the use of chemical pesticide, can also be cost-saving for agriculture production, reduce environmental pollution and raise the management level, and therefore the present invention has wide market application foreground.
Accompanying drawing explanation
Fig. 1 is the PCR detected result schematic diagram of part transgene tobacco genomic dna in the present invention, wherein: Marker is DL2000 DNA Marker (Dalian is precious biological), by 2,000bp, 1,000bp, 750bp, 500bp, 250bp and six DNA fragmentation compositions of 100bp; Positive control is plasmid pMD-18T- lrGLP2for the PCR reaction of template; WT is that the total DNA of non-transgene tobacco (wild-type) is the PCR that template is carried out;
Fig. 2 is in the present invention in some positive transgene tobacco lrGLP2the expression analysis result figure of transcriptional level, wherein: Marker is that DL2000 DNA Marker(Dalian is precious biological); WT is that the total RNA of non-transgene tobacco reverses the PCR product that cDNA is template; Positive control is plasmid pMD-18T- lrGLP2for the PCR product of template;
Fig. 3 is in the present invention lrGLP2the fungistatic effect figure of transgene tobacco extracorporeal antifungal activity, wherein: the fungi in a, b, c, d diagram is respectively grape seat chamber bacterium, Phomopsis fungi, Fusarium oxysporum, Botrytis cinerea; WT is the total protein of wild-type tobacco; CK is blank, without albumen contrast (for extracting the damping fluid of albumen).
Embodiment
Below by embodiment, the present invention is described in further detail, but content of the present invention is not limited to this, method all operations according to a conventional method if no special instructions in the present embodiment, the conventional reagent of agents useful for same employing if no special instructions or the according to a conventional method reagent of configuration.
Embodiment 1: lrGLP2full-length gene clone and sequential analysis
Inoculate lilium regale wilson with Fusarium oxysporum, extract total RNA with the root after inoculation 24 h,, proceed in centrifuge tube the root grind into powder of the lilium regale wilson of processing with liquid nitrogen, employing guanidine isothiocyanate method extracts total RNA.Adopt reversed transcriptive enzyme M-MLV(promega) synthetic cDNA the first chain take total RNA as template, reaction system and operating process are: get 5 μ g Total RNA, add successively 50 ng oligo(dT),, DEPC water to reaction volume is 12.5 μ L; After mixing, after 70 ℃ of heat denatured 5min rapidly at cooled on ice 5 min, then add successively 4 μ L 5 × First-stand buffer, 2 μ L dNTP(2.5mM each) 0.5 μ L RNasin(200U), 1 μ L M-MLV(200U), mix and centrifugal in short-term, 42 ℃ of temperature are bathed 1.5 h, take out rear 70 ℃ of heating 10 min, termination reaction.CDNA the first chain is synthetic to be placed on-20 ℃ and to save backup.
Take the first chain cDNA of synthesizing as template, amplifying target genes lrGLP2, upstream and downstream primer sequence used is respectively 5 ' GGATCCGCAGTGGTATCAACGCAGAGTACA3 ', 5 ' GAATTCATCCATCAGAACT TAGCCTGGAGC3 '.Adopt Advantage tM2 PCR Enzyme (Clontech) amplify goal gene, PCR reaction conditions: 95 ℃ of 1 min; 95 ℃ of 30 s, 62 ℃ of 30 s, 72 ℃ of 60 s, 30 circulations; 72 ℃ of 2min.Reaction system (20 μ L) is 2 μ L cDNA, 2 μ L 10 × Advantage 2 PCR Buffer, 0.5 μ L 50 × dNTP Mix(10mM each), 0.2 μ L forward primer (10 μ M), 0.2 μ L reverse primer (10 μ M), 0.2 μ L Advantage2 PCR Polymerase Mix, 14.9 μ L PCR-Grade water.After PCR finishes, get 5 μ L for agarose gel electrophoresis, detect specificity and the size of amplified production.
Because PCR product only has a DNA band, therefore directly PCR product is carried out to TA clone, the test kit using is the precious biology in pMD18-T vector kit(Dalian), reaction system and operating process are: get 1.5 μ L PCR products, add successively 1 μ L pMD18-T vector(50 ng/ μ L) and 2.5 μ L 2 × Ligation solution I, mix and be placed in 16 ℃ of reaction overnight.Adopt heat shock conversion method that connection product is proceeded in bacillus coli DH 5 alpha.The LB solid medium screening positive clone that use contains penbritin (ampicillin, Amp).Select several single bacterium colonies, shake after bacterium with amplification lrGLP2special primer identify multiple clone site insert lrGLP2clone.Identified clone is checked order, final acquisition lrGLP2full-length cDNA is 921bp, by NCBI ORF finder(http: //www.ncbi.nlm.nih.gov/gorf/gorf.html) analyze and find its opening code-reading frame that comprises a 654bp (seeing sequence table), lrGLP2encode one and contain 217 amino acid whose Protein L rGLP2, its molecular weight is about 23.35KDa, and iso-electric point is 6.16, contains 1 cysteine residues (C), is positioned at the 46th, and monomeric protein forms without disulfide linkage.Analyze by bioinformatics software SignalP 4.1 lrGLP2the protein sequence of coding, detects it and whether has N end signal peptide, and result shows the signal peptide of 1st ~ 21 amino acids composition LrGLP2 of this albumen, and possible shearing site is between 21st ~ 22 amino acid.The signal peptide that is positioned at protein N-end is the typical marks of secretory protein, illustrates that LrGLP2 is a kind of secretory protein.
Embodiment 2: plant expression vector construction
Adopt extraction agent box (the raw work in Shanghai) the extraction insertion from intestinal bacteria in a small amount of SanPrep pillar plasmid DNA lrGLP2plasmid pMD-18T- lrGLP2and the plasmid of plant expression vector pCAMBIA2300s, get 1 μ L for agarose gel electrophoresis to detect the integrity of the plasmid that extracted and concentration just.Use restriction enzyme ecorI (TaKaRa) and bamhI(TaKaRa) respectively to plasmid pMD-18T- lrGLP2carry out double digestion (100 μ L system) with pCAMBIA2300s, reaction system and operating process are: get 20 μ L pMD-18T- lrGLP2with pCAMBIA2300s plasmid, add 10 μ L 10 × K buffer, 5 μ L successively ecoRI, 5 μ L bamhI, 60 μ L ddH 2o, centrifugal in short-term after mixing, be placed in 37 ℃ of reaction overnight.All enzymes are cut to product point and in sepharose, carry out electrophoresis, then right lrGLP2gene fragment and pCAMBIA2300s carrier large fragment are carried out respectively glue recovery, whole process is used SanPrep pillar DNA glue to reclaim test kit (the raw work in Shanghai), get 1 μ L recovery product and detect the size and the concentration that reclaim fragment by agarose gel electrophoresis, be placed in-20 ℃ and save backup.
Utilize T4 DNA Ligase(TaKaRa), by what reclaim lrGLP2dNA fragmentation and pCAMBIA2300s carrier segments couple together, and reaction system (20 μ L) and operating process are: get 10 μ L lrGLP2dNA fragmentation adds 2 μ L pCAMBIA2300s carrier DNAs, 2 μ L 10 × T4 DNA Ligase Buffer, 1 μ L T4 DNA Ligase, 5 μ L ddH successively 2o, centrifugal in short-term after mixing, be then placed in 16 ℃ of water-bath reaction overnight.Then adopt heat shock conversion method that connection product is proceeded in bacillus coli DH 5 alpha, with the solid medium screening positive clone that contains 50 mg/L kantlex (kanamycin, Km).Select single bacterium colony and shake bacterium, use amplification take bacterium liquid as template lrGLP2special primer carry out PCR, pick out lrGLP2the clone who is successfully connected with pCAMBIA2300s, if the bacterial strain detecting is positive, adds glycerine and is placed in-80 ℃ to save backup.
Adopt the pCAMBIA2300s in (the raw work in the Shanghai) extraction of SanPrep pillar plasmid extraction test kit the above-mentioned intestinal bacteria of purifying -LrGLP2plasmid, uses frozen-thawed method by the plant expression vector pCAMBIA2300s-of above-mentioned structure subsequently lrGLP2proceed in prepared agrobacterium tumefaciens lba4404 competent cell, operation steps is: get 0.2 μ g pCAMBIA2300s- lrGLP2plasmid adds in the centrifuge tube that contains 200 μ L competent cells, mixes gently rear ice bath 5 min, then proceeds to freezing 1 min in liquid nitrogen, then be placed in rapidly 37 ℃ of water-bath 5 min, ice bath 2 min immediately afterwards, add 800 μ L LB liquid nutrient mediums, 28 ℃ of shaking culture 4 h.Agrobacterium after activation is applied on the LB solid medium that contains 50 mg/L Km to 28 ℃ of static cultivations.Select single bacterium colony and shake bacterium, with amplification lrGLP2auele Specific Primer carry out PCR, detect pCAMBIA2300s- lrGLP2whether proceed in Agrobacterium.For positive colony, add glycerine to be placed on-80 ℃ and save backup.
Embodiment 3: agriculture bacillus mediated Genetic Transformation in Higher Plants and transgenic plant screening
The transgene receptor of this experiment is tobacco, by 75% alcohol-pickled 30s for tobacco seed, with the HgCl with 0.1% after sterilized water washing 2soak 8 min, and then wash several times with sterilized water, be seeded on 1/2MS substratum, 28 ℃ of dark 5-8 d that cultivate, go to illumination box (25 ℃, 16 h/d illumination) after germination, monthly use MS substratum subculture once later.
From-80 ℃ of refrigerators, take out the pCAMBIA2300s-that contains preserving lrGLP2the Agrobacterium LBA4404 bacterial classification of plasmid, is inoculated in the LB liquid nutrient medium that 5 mL contain 50 mg/L Km and 20 mg/L Rifampins, and 28 ℃ are cultured to substratum muddiness.Draw the bacterium liquid of 1 mL muddiness and coat on the LB solid medium that contains 50 mg/L Km, cultivate 48 h for 28 ℃.Agrobacterium on LB solid medium is scraped in the MGL liquid nutrient medium that is inoculated in right amount the Syringylethanone that is attached with 20 mg/L, 28 ℃ of shaking culture 3 h are with activation Agrobacterium.
Get tobacco aseptic seedling leaf and be cut into 1 cm 2the leaf dish of left and right, is soaked in above-mentioned containing in the MGL liquid nutrient medium that activates Agrobacterium completely, and immerged time is 15 min.Blot the bacterium liquid of leaf panel surface with aseptic filter paper, leaf dish is placed in and on common substratum, carries out incubated at room temperature.The common substratum of Transformation of tobacco is MS+0.02 mg/L 6-BA+2.1 mg/L NAA+30 g/L sucrose+6 g/L agar, and 22 ℃ without cultivating altogether 2 d under optical condition.
Leaf dish after common cultivation is forwarded to and is added with seedling differentiation in antibiotic MS screening culture medium, simultaneously screening transgenic plant.Tobacco screening and culturing joint is MS+0.5 mg/L 6-BA+0.1 mg/L NAA+30g/L sucrose+6 g/L agar+50 mg/L Km+200 mg/L cephamycins (cefotaxime sodium salt, Cef); When screening and culturing, culturing bottle is transferred to illumination box and cultivates (25 ℃, 16 h/d illumination, 8 h/d dark), after sprouting, use the MS substratum succeeding transfer culture that contains 50 mg/L Km and 200 mg/L Cef, because of tobacco callus differentiation rate higher, therefore need to further screen regeneration plant, tobacco regrowth is moved on the MS substratum that contains 50 mg/L Km it is taken root, finally select the good regrowth of taking root to do further detection.
Adopt CTAB method to extract the genomic dna of transgenic tobacco plant blade, the genomic dna of extraction is got to 1 μ L and detect its integrity and concentration by agarose gel electrophoresis, use and increase as template take the genomic dna of transfer-gen plant lrGLP2special primer carry out PCR, after PCR finishes, get 8 μ L products for agarose gel electrophoresis to detect positive transfer-gen plant, the amplification of part transgenic tobacco plant as shown in Figure 1, lrGLP2transgene tobacco screens the positive transfer-gen plant of 35 strains altogether.
Embodiment 4: lrGLP2expression analysis and transfer-gen plant anti-mycotic activity are analyzed
The tender leaf of getting positive transgenosis individual plant and non-transgenic tobacco (wild-type, WT) extracts total RNA, and reverse transcription generates cDNA the first chain, and as amplification for template lrGLP2special primer carry out PCR, according in the each transgenosis individual plant of PCR interpretation of result lrGLP2the expression of transcriptional level, method and the step of total RNA extraction and RT-PCR are in the same manner as in Example 1, after PCR finishes, get 5 μ L for agarose gel electrophoresis, and the detected result of part individual plant is as shown in Figure 2.Detect altogether in 19 strain transgenosis individual plants lrGLP2have expression at transcriptional level, these individual plants be numbered 1～19.
Several pathogenic fungies that laboratory is preserved are inoculated in PDA solid medium (200 g/L potatos, 15 g/L agar, 20 g/L glucose) on, 28 ℃ of dark cultivations, in the time that being about 2～3cm, adds diameter albumen until colony growth, analyze transfer-gen plant extracorporeal antifungal activity, have 5 kinds for examination fungi: Phomopsis ( phomopsissp.) fungi, grape seat chamber bacterium ( botrosphaeria dothidea), Fusarium oxysporum ( fusarium oxysporum), Alternariaspp ( alternariasp.), Botrytis cinerea ( botrytis cinerea).
For the albumen that prevents that other living contaminants from extracting, whole vegetable-protein leaching process is all aseptic techniques, first get 1 g transgene tobacco individual plant (numbering is respectively 1,2,3,8,12) or wild-type blade and put into mortar, add 1 mL protein extract (1M NaCl, 0.1M sodium acetate, 1% PVP, pH6), fully grind.Proceed in 1.5 mL centrifuge tubes, mix rear 4 ℃ of hold over night.4 ℃ of centrifugal 30 min (12, 000 g/min), get supernatant in 1.5 new mL centrifuge tubes, and get appropriate with uv-spectrophotometric instrument mensuration total protein concentration, the total protein concentration of transgenosis and wild-type plant is adjusted to 0.2 μ g/ μ L, then getting respectively 20 μ L drips on the aseptic filter paper of each fungi culture medium, on the flat board of each fungi except adding the total protein of different transgenic tobacco plants, total protein and the blank (CK of parallel interpolation wild-type tobacco simultaneously, extract albumen solution used), cultivate the situation of observing afterwards the antibacterial fungal growth of each processing for several days for 28 ℃, and evaluate accordingly lrGLP2the extracorporeal antifungal activity of transgene tobacco, result as shown in Figure 3, lrGLP2transgene tobacco albumen has very strong inhibition activity to the growth of grape seat chamber bacterium and Phomopsis fungi, and the growth of Fusarium oxysporum and Botrytis cinerea is also had to obvious restraining effect.
Sequence table (SEQ ID)
<110> Kunming University of Science and Technology
<120> lilium regale wilson class is sprouted fibroin gene lrGLP2and application
<160> 4
<170> PatentIn version 3.5
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<211> 921
<212> DNA
<213> LiliumregaleWilson
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atggggacct ttgagaaatt cttcccgatt gctaaaatat aacgcacaca tggctaccca 60
ctacttcttc tggctcgcca tccttgccct agcctcctcc cattcaatgg cttctgatcc 120
tgactcactg caggatttct gtgtggctga ccgaaactcc caagtgattg tgaatggatt 180
agtctgcaag gacccaaagc aagcccaacc tgctgatttt ttcttccccg gactgaacca 240
gccagggagc acaaccaacc agctggggtc taacgtcact cttgtcagcg cggcaaacct 300
tccggggctc aacacccttg gaatctctgt ggctcgcctg gactttgcac cgtatggcct 360
cattccccct cactaccatc cacgtgcgac cgagatcctg acattgctgg agggtactct 420
ctacgttggt tttgtcactt ccttccctga cttccgtctc attagcaaga tcctcaaggc 480
cggtgatgtc tttgtcttcc ccaaaggact cgtccacttc cagtttaatc aggacggcac 540
caaacctgca gtttccctct caggtttgag cagccagaac ccaggactgg tcactgtagc 600
taatgccgtt ttcggatcta acccgcctat ctgggatgac gttctggcca aggccttcca 660
gttggacaag aagacagttg acgggctcca ggctaagttc tgatggatta ctcagttgtg 720
atatcaccta ataaatatag atgtttgtgg actatagtgt gtaataaaca aactgatgtt 780
ttcatgtttc tagatcatta tttagattac acgtaaagtt gctcggctat tcaaatttct 840
ccttccatct attatatata tattctccgc agaattcaat aaaattcatt ccatcaccaa 900
aaaaaaaaaa aaaaaaaaaa a 921
<210> 2
<211> 217
<212> PRT
<213> LiliumregaleWilson
<400> 2
Met Ala Thr His Tyr Phe Phe Trp Leu Ala Ile Leu Ala Leu Ala Ser
1 5 10 15
Ser His Ser Met Ala Ser Asp Pro Asp Ser Leu Gln Asp Phe Cys Val
20 25 30
Ala Asp Arg Asn Ser Gln Val Ile Val Asn Gly Leu Val Cys Lys Asp
35 40 45
Pro Lys Gln Ala Gln Pro Ala Asp Phe Phe Phe Pro Gly Leu Asn Gln
50 55 60
Pro Gly Ser Thr Thr Asn Gln Leu Gly Ser Asn Val Thr Leu Val Ser
65 70 75 80
Ala Ala Asn Leu Pro Gly Leu Asn Thr Leu Gly Ile Ser Val Ala Arg
85 90 95
Leu Asp Phe Ala Pro Tyr Gly Leu Ile Pro Pro His Tyr His Pro Arg
100 105 110
Ala Thr Glu Ile Leu Thr Leu Leu Glu Gly Thr Leu Tyr Val Gly Phe
115 120 125
Val Thr Ser Phe Pro Asp Phe Arg Leu Ile Ser Lys Ile Leu Lys Ala
130 135 140
Gly Asp Val Phe Val Phe Pro Lys Gly Leu Val His Phe Gln Phe Asn
145 150 155 160
Gln Asp Gly Thr Lys Pro Ala Val Ser Leu Ser Gly Leu Ser Ser Gln
165 170 175
Asn Pro Gly Leu Val Thr Val Ala Asn Ala Val Phe Gly Ser Asn Pro
180 185 190
Pro Ile Trp Asp Asp Val Leu Ala Lys Ala Phe Gln Leu Asp Lys Lys
195 200 205
Thr Val Asp Gly Leu Gln Ala Lys Phe
210 215
<210> 3
<211> 30
<212> DNA
<213> artificial sequence
<400> 3
ggatccgcag tggtatcaac gcagagtaca 30
<210> 4
<211> 30
<212> DNA
<213> artificial sequence
<400> 4
gaattcatcc atcagaactt agcctggagc 30

Claims (3)

1. a lilium regale wilson class is sprouted fibroin gene lrGLP2, it is characterized in that: described class is sprouted fibroin gene lrGLP2nucleotide sequence as shown in SEQ ID NO:1, this full length gene is 921bp, has open reading frame, the 5 ' non-translational region of 49bp and the 3 ' non-translational region of 218bp of 654bp, coding protein of aminoacid sequence as shown in SEQ ID NO:2.
2. lilium regale wilson class according to claim 1 is sprouted fibroin gene lrGLP2improve tobacco to the application in grape seat chamber bacterium, Fusarium oxysporum, Botrytis cinerea resistance.
3. lilium regale wilson class according to claim 2 is sprouted fibroin gene lrGLP2application, it is characterized in that the concrete operations of the fungus resistant that improves tobacco are as follows:
(1) above-mentioned class is sprouted to fibroin gene and be connected with plant expression vector pCAMBIA2300s, build plant overexpression vector;
(2) recombinant vectors of above-mentioned structure is proceeded in target plant by Agrobacterium tumefaciens mediated;
(3) the resistance marker screening transformant to have on recombinant vectors T-DNA, and obtain real transfer-gen plant by polymerase chain reaction, inoculation specific pathogen fungi, analyze the inhibition activity of transgenic plant albumen to fungal growth, finally filter out the transfer-gen plant that fungus resistant is obviously strengthened.
CN201310143928.0A 2013-04-24 2013-04-24 Lilium regale germin-like protein gene LrGLP2 and application thereof Expired - Fee Related CN103194457B (en)

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Families Citing this family (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104131014B (en) * 2014-08-06 2016-02-24 昆明理工大学 Lilium regale wilson class sprouts the application of fibroin gene LrGLP1
CN104152465B (en) * 2014-08-13 2017-01-11 昆明理工大学 Lilium regale cytochrome b5 gene LrCyt-b5 and application thereof
CN104131015B (en) * 2014-08-13 2016-05-25 昆明理工大学 The application of a kind of lilium regale wilson pathogenesis-related proteins 10 gene LrPR10-5
CN106432452A (en) * 2016-12-12 2017-02-22 中国热带农业科学院环境与植物保护研究所 Rubber tree disease-resistant related protein GLPs and coding gene and application thereof
CN108707610B (en) * 2018-05-04 2021-01-05 昆明理工大学 Notoginseng defensein antibacterial peptide genePnDEFL1And applications
CN114835789B (en) * 2022-05-25 2023-12-22 河南科技学院 Wheat powdery mildew resistance related protein TaGLP-7A, and coding gene and application thereof

Non-Patent Citations (12)

* Cited by examiner, † Cited by third party
Title
BvGLP1过表达增强了转基因小麦对根腐病的抗性;党良等;《作物学报》;20130228;第39卷(第2期);368－372 *
EU975676.1;Lu Y.P.等;《EMBL-EBI》;20081216;1－3 *
EY412924.1;Low E.T.L.等;《EMBL-EBI》;20110310;1－3 *
JO093866.1;Shearman J.R.等;《EMBL-EBI》;20130313;1－3 *
Low E.T.L.等.EY412924.1.《EMBL-EBI》.2011,1－3.
Lu Y.P.等.EU975676.1.《EMBL-EBI》.2008,1－3.
Shearman J.R.等.JO093866.1.《EMBL-EBI》.2013,1－3.
党良等.BvGLP1过表达增强了转基因小麦对根腐病的抗性.《作物学报》.2013,第39卷(第2期),368－372.
唐源江 等.拟南芥GLP13基因在植物抗氧化胁迫响应中的作用.《植物学报》.2011,第46卷(第2期),147－154.
拟南芥GLP13基因在植物抗氧化胁迫响应中的作用;唐源江 等;《植物学报》;20110228;第46卷(第2期);147－154 *
王俊美 等.白粉菌诱导的小麦类萌发素蛋白的克隆、定位及表达分析.《中国农业科学》.2009,第42卷(第9期),3104－3111.
白粉菌诱导的小麦类萌发素蛋白的克隆、定位及表达分析;王俊美 等;《中国农业科学》;20090930;第42卷(第9期);3104－3111 *

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