CN103320448A - Lilium regle bZIP transcription factor LrbZIP1 and application - Google Patents
Lilium regle bZIP transcription factor LrbZIP1 and application Download PDFInfo
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- CN103320448A CN103320448A CN2013102583698A CN201310258369A CN103320448A CN 103320448 A CN103320448 A CN 103320448A CN 2013102583698 A CN2013102583698 A CN 2013102583698A CN 201310258369 A CN201310258369 A CN 201310258369A CN 103320448 A CN103320448 A CN 103320448A
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Abstract
The inventing discloses a Lilium regle bZIP transcription factor LrbZIP1. According to the invention, the nucleotide sequence of the gene LrbZIP1 is as shown in SEQ ID No:1, and the gene LrbZIP1 encodes a protein of a nucleotide sequence as shown in SEQ ID No:2. Functional genomics related technical research proves that LrbZIP1 has a function for improving the antifungal property of plants, after the antifungal LrbZIP1 gene is constructed in a plant expression vector and transferred to tobacco for overexpression, the result shows that the transgenic tobacco plant has strong in-vitro antifungal activity, so the experiment shows that the LrbZIP1-overexpressed transgenic tobacco has an obvious inhibiting effect on growth of various fungus such as ascomycetes and fusarium oxysporum.
Description
Technical field
The present invention relates to molecular biology and genetically engineered correlation technique research field, particularly a kind of lilium regale wilson bZIP transcription factor gene with anti-mycotic activity
LrbZIP1And use.
Background technology
Plant diseases is to be very stubborn problem, especially a fungal disease in agriculture production, accounts for 80% of the total disease of plant, is having a strong impact on the yield and quality of farm crop.Although traditional pest control method has been obtained certain effect, but it mainly relies on traditional breeding way to cultivate resistant variety and uses chemical pesticide, or take the cropping systems such as crop rotation, all there is drawback more or less in these methods, as use waste time and energy, the residual height of chemical pesticide and easily long etc. to environment, breeding cycle, so the thorough problem of the method for tradition control Plant diseases.Along with foundation and the development of recombinant DNA technology, utilize the genetic engineering technique new variety that cultivate plants to obtain first-stage success with the reply fungal disease, and be expected to fundamentally solve the fungal disease problem.
After being subject to the external environment such as pathogenic bacteria when plant and changing hormesis, can produce a series of signal conductive process in its body, the expression of the relevant resistant gene of inducing plant defence, as when being subject to the cause of disease invasion and attack, plant can be activated associated transcription factor by a series of signal conduction, distinguished sequence and with it combination on the transcription factor identification resistance related gene promoter region, start the transcriptional expression of target gene, and then the biochemical reactions of inducing the series of defence pathogenic bacteria to invade, make plant produce disease-resistant characteristic.According to the different characteristics of the structural domain of being combined with DNA, transcription factor can be divided into AP2/EREBP, bZIP, WRKY, MYB and the types such as NAC, Zinc-finger.As a class transcription factor that extensively exists in the eukaryote, the defense response of alkaline leucine zipper (Basic leucine zipper, bZIP) transcription factor and plant is closely related.The bZIP transcription factor be distribute in the eukaryote transcription factor the most extensively, a most conservative proteinoid, formed in conjunction with territory and a leucine zipper dimeric structure territory by a DNA, be one of gene family larger in the transcription factor, almost in all eukaryotes, all have the albumen that contains the bZIP structural domain.At present, Arabidopis thaliana (
Arabidopsis thaliana), soybean (
Glycine max), paddy rice (
Oryza sativa) find a large amount of bZIP transcription factor genes in the genome.BZIP transcription factor gene family member different amts (the Landschulz WH that different plants contain, Johnson PF, McKnight SL. The leucine zipper:a hypothetical structure common to a new class of DNA binding proteins. Science, 1988,240:1759-1764).According to basic domain and other conservative structural domain, 77 bZIP class transcription factor gene family members in the Arabidopis thaliana are divided into 10 subfamilies such as A, B, C, D, E, F, G, H, I and S.In addition, finding in the soybean gene group has 131 bZIP class transcription factor gene family members, and finding in the rice genome has 89 bZIP transcription factors, equally also is divided into 10 subfamilies.Generally has similar feature between same class subfamily member, such as the size of leucine zipper.The bZIP transcription factor participates in regulate several biological processes, comprising seed maturity, optical signal, flower development, pathogenic bacteria defence and to the response of multiple environment stress etc.
NPR1(Nonexpressor of pathogenesis related gene 1) gene is a key gene of regulating plant disease resistance, to Systemic Acquired Resistance In Plants (Systemic acquired resistance, SAR) and inducible system resistance (Induced systemic resistance, ISR) play crucial regulating and controlling effect.By yeast two-hybrid experiment (yeast two-hybrid system, Y
2H) verified
NPR1By with bZIP class transcription factor family member TGA(Thymine guanine adenine) thereby protein-interacting regulate
PR-1The expression of gene.In Arabidopis thaliana, TGA albumen can in conjunction with
NPR1Whitfield's ointment SA(salicylic acid on the promotor) functional element improves
PR-1Expression.After plant was subject to pathogenic bacteria and attacks, the content that starts the signaling molecule SA of disease-resistant signal transduction in the plant materials improved, and NPR1 albumen becomes monomer by polymer, entered in the nucleus and the TGA protein-interacting, and then regulated
PRThe expression of gene, make the disease resistance of plant strengthen (Zhou JM, Trifa Y, Silva H, Pontier D, Lam E, Shah J, Klessig DF. NPR1 differentially interacts with members of the TGA/OBF family of transcription factors that bind an element of the
PR-1Gene required for induction by salicylic acid. Mol Plant Microbe Interact, 2000,13:191-202).Tobacco (
Nicotiana tabacum) in overexpression sudden change with the Arabidopis thaliana that can not be combined with dna sequence dna
TGA2Gene, the late phase responses genoid
PR1, PR2And
PR3Expression increase, and to the wildfire bacterium (
Pseudomonas syringaePv.
Tabaci) resistance strengthen, show that TGA2 plays negative regulation effect (Pontier RD in genetic expression and defensive raction, Miao ZH, Lam E. Trans-dominant suppression of Plant TGA factors reveals their negative and positive roles in plant defense responses. Plant Journal, 2001,27:529-538).In addition, make paddy rice by PCR method
RTGA2.1Gene DNA sports proline(Pro) in conjunction with two alanine residues in the territory, and formation can not be in conjunction with the mutant of specific dna sequence
RTGA2.2, overexpression
RTGA2.2The paddy rice of gene or utilize the double chain RNA mediate paddy rice endogenous
RTGA2.1Gene silencing, can improve paddy rice to bacterial leaf spot pathogenic bacteria (
Xanthomanos oryzaePV.
Oryzae) resistance, show paddy rice
RTGA2.1In the defense response to bacterial pathogens, play negative regulation effect (Fitzgerald HA, Canlas PE, Chern MS, Ronald PC. Alteration of TGA factor activity in rice results in enhanced tolerance to
Xanthomanos oryzaePV.
Oryzae. Plant Journal, 2005,43:335-347).
When plant was subjected to the pathogenic bacteria invasion and attack or meets with other environment stresses, the aerobic repiration meeting made plant produce excessive active oxygen (reactive oxygen species, ROS), and therefore effective antioxidant system seems extremely important under exceedingly odious envrionment conditions.During salt stress, the overexpression Tamarix hispida (
Tamarix hispida)
ThbZIP1In the transgene tobacco of gene, the increased activity of superoxide-dismutase (SOD) and peroxidase (POD), and the increase of the content of soluble sugar and soluble proteins show
ThbZIP1Can improve the resistance to salt stress by the multiple physiological pathway of regulation and control, in addition,
ThbZIP1Can increase the removing to active oxygen, the biosynthesizing (Wang YC, Gao C, Liang Y, the et al. A novel that induce generation, increase soluble proteins of promotion solubility permeate agent
BZIPGene from
Tamarix hispidaMediates physiological responses to salt stress in tobacco plants [J]. Journal of Plant Physiology, 2010,167:222-230).From capsicum (
Capsicum annuum) middle separation
CAbZIP1Gene, change overexpression in the Arabidopis thaliana over to, the result shows that transfer-gen plant strengthens (Lee SC to the resistance of bacterial spot of tomato bacterium (tomato bacterial spots pathogen), Choi HW, Hwang IS, Choi du S, Hwang BK. Functional roles of the pepper pathogen induced bZIP transcription factor
CAbZIP1, in enhanced resistance to pathogen infection and environmental stresses. Planta, 2006,224:1209-1225).Change capsicum over to
CAbZIP1The Arabidopis thaliana of gene in whole growth and development process, is compared with the non-transgenic strain, and the drought resisting of plant and salt resistance improve, and overexpression
CabZIP1The Arabidopis thaliana of gene is removed enzyme by regulation and control ROS, such as peroxidase and catalase (CAT), eliminates the too much active oxygen in the plant materials, and plant is strengthened the resistance of oxidative stress.
BZIP transcription factor gene of the present invention
LrbZIP1From lilium regale wilson (
Lilium regaleWilson).Lilium regale wilson has another name called regallity, per nnial herb, the lily endemic species of China.Only be distributed in the river valley of west, river Minjiang River Basin height above sea level 800～2700m in the rock seam on hill-side, have extremely strong disease resistance.
Summary of the invention
The purpose of this invention is to provide a kind of full-length gene that clone's acquisition coding has the bZIP transcription factor of anti-mycotic activity from lilium regale wilson
LrbZIP1, the bZIP transcription factor gene
LrbZIP1Nucleotide sequence is as described in the SEQ ID NO:1, this gene cDNA full length sequence is 979bp, comprise the open reading frame of a 429bp, the 5 ' non-translational region of 305bp, the 3 ' non-translational region of 245bp, the protein of coding aminoacid sequence shown in SEQ ID NO:2.
Gene among the present invention
LrbZIP1The coding region be the nucleotide sequence shown in the 306-734 position among the sequence table SEQ ID NO:1.
The global cDNA fragment of an antimycotic genes involved of separating clone lilium regale wilson of the present invention, utilize and Agrobacterium tumefaciens mediated goal gene is changed in the 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 plants lays the foundation, and the contriver with this unnamed gene is
LrbZIP1
The bZIP transcription factor is the extensive and the most conservative proteinoid that distributes in all eukaryote transcription factors, plant bZIP transcription factor combines by the special cis functional element with many defense response genes upstream promoters zone, the expression of regulation and control goal gene, bZIP class transcription factor identification core sequence is the cis-acting elements of ACGT, such as the TACGTA(A box), the CACGTG(G box), the GACGTC(C box).The bZIP transcription factor by with the plant disease-resistant key controlling gene
NPR1Interact and adjusting pathogenesis-related proteins (pathogenesis-related protein, PR) expression of gene, can also remove by regulation and control ROS the activity of enzyme, such as peroxidase and catalase etc., eliminate the too much active oxygen that forms in the plant materials, plant is strengthened the resistance of oxidative stress.Therefore, the bZIP transcription factor is invaded and is tackled in other environment stress process at plant opposing pathogenic fungi and plays an important role.
The present invention relates to separate and comprise
LrbZIP1Dna fragmentation and identify its function, the plant with this gene fragment has the phenotype of the specific fungal attack of opposing to a certain extent, wherein said dna fragmentation carries out sequential analysis to this gene shown in sequence table SEQ ID NO:1, find
LrbZIP1Full-length cDNA is 979bp, comprises the open reading frame (ORF) of a 429bp, the 5 ' non-translational region (untranslated region, UTR) of 305bp and the 3 ' UTR of 245bp, and wherein one of ORF coding has 142 amino acid whose protein.
LrbZIP1Proteins encoded has the conserved domain of bZIP albumen, and the BLASTp result for retrieval shows
LrbZIP1The similarity of the protein of coding and Arabidopis thaliana, capsicum, soybean, corn (Zea mays), paddy rice bZIPs transcription factor is about 50%, shows that it belongs to the bZIP transcription factor in the lilium regale wilson.Protein shown in the overexpression sequence table SEQ ID NO:2 can strengthen tobacco to the resistance of grape seat chamber bacterium, Phomopsis fungi, Fusarium oxysporum, Alternariaspp.
Another purpose of the present invention is with lilium regale wilson bZIP transcription factor gene
LrbZIP1Be applied in and improve tobacco in grape seat chamber bacterium, Phomopsis fungi, Fusarium oxysporum, the Alternariaspp resistance, concrete operations are as follows:
(1) adopts amplification
LrbZIP1Special primer, from the lilium regale wilson root of inoculation behind the Fusarium oxysporum, extract total RNA, (reverse transcription-polymerase chain reaction, RT-PCR) amplifies by reverse transcription-polymerase chain reaction
LrbZIP1Full length coding region, then be connected on the pMD-18T carrier, obtain to have the clone of goal gene through order-checking;
(2) use restriction enzyme
BamHI and
EcoThe RI enzyme is cut pMD-18T-
LrbZIP1Carrier obtains the goal gene fragment by the glue recovery, and with same endonuclease digestion plant expression vector pCAMBIA2300s, glue reclaims and obtains required carrier large fragment, institute is obtained again
LrbZIP1Gene fragment is connected with the pCAMBIA2300s fragment, makes up plant overexpression vector, afterwards constructed recombinant vectors is expressed by Agrobacterium tumefaciens mediated changing in the tobacco;
(3) the resistance marker screening transformant to have on the recombinant vectors T-DNA, and detect by PCR and RT-PCR and to obtain real transfer-gen plant, analyze transgenic plant albumen active to the inhibition of fungal growth, filter out at last 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, cultivates the deficiency that disease-resistant plants can overcome traditional breeding method by genetic engineering means, and not only breeding cycle shortens, and simple to operate, easily obtains the high resistance material.The present invention is from lilium regale wilson
LrbZIP1Gene can strengthen plant to the resistance of fungi, and this gene is imported in the tobacco, can produce new variety and novel material with fungus resistant.The importance of utilizing genetic engineering technique cultivation resistance plant kind and material to have obvious advantage and do not replace.It not only can be provided convenience for scale operation crop, flowers etc., reduces in a large number the use of chemical pesticide, can also save cost for agriculture production, reduce environmental pollution, so the present invention has wide market application foreground.
Description of drawings
Fig. 1 is the present invention
LrbZIP1The PCR detected result of transgene tobacco genomic dna, among the figure: Marker is DL2000 DNA Marker (Dalian is precious biological); Positive control is plasmid pMD-18T-
LrbZIP1PCR knot product for template; WT is that the total DNA of non-transgenic tobacco (wild-type) is the product of template PCR;
Fig. 2 is that the present invention is positive
LrbZIP1In the transgene tobacco
LrbZIP1The expression analysis of transcriptional level is figure as a result; Among the figure: Marker is that DL2000 DNA Marker(Dalian is precious biological); WT is that the total RNA reverse transcription of non-transgene tobacco cDNA is the PCR product of template; Positive control is plasmid pMD-18T-
LrbZIP1PCR product for template;
Fig. 3 is the present invention
LrbZIP1The fungistatic effect schematic diagram of transgene tobacco extracorporeal antifungal activity; Fungi among the figure among a, b, c, the d is respectively grape seat chamber bacterium, Phomopsis fungi, Fusarium oxysporum, Alternariaspp; WT is the total protein of wild-type tobacco; CK is blank, namely without albumen contrast (being used for extracting the damping fluid of albumen).
Embodiment
Below by drawings and Examples the present invention is described in further detail, but protection domain of the present invention is not limited to described content.
Embodiment 1:
LrbZIP1Full-length gene clone and sequential analysis
Inoculate lilium regale wilson with Fusarium oxysporum, extract total RNA with the root behind inoculation 24 h, the root grind into powder of the lilium regale wilson that will process with liquid nitrogen, then change in the centrifuge tube, adopt guanidine isothiocyanate method to extract 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), 2 μ L dNTP(2.5mM each), DEPC water to reaction volume is 14.5 μ L; Behind the mixing, behind 70 ℃ of heat denatured 5min rapidly at cooled on ice 5min, then add successively 4 μ L, 5 * First-stand buffer, 0.5 μ L RNasin(200U), 1 μ L M-MLV(200U), mixing is also 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 synthetic chain cDNA as template, amplifying target genes
LrbZIP1, used upstream and downstream primer sequence is respectively 5 ' GGATCCCGTCCTTCTCTACTGGTTCTATGC3 ' and 5 ' GAATTCCATGAAACTCAAAC TAGATCACTGG3 '.Adopt Advantage
TM2 PCR Enzyme(Clontech) amplify goal gene; PCR reaction conditions: 95 ℃ of 1 min; 95 ℃ of 30 s, 58 ℃ of 30 s, 72 ℃ of 50 s, 30 circulations; 72 ℃ of 5 min; Reaction system (10 μ L) is 1 μ L cDNA, 1 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 Advantage, 2 PCR Polymerase Mix, 6.9 μ L PCR-Grade water; After PCR finishes, get 5 μ L and be used for agarose gel electrophoresis, in order to specificity and the size that detects amplified production.
Resulting PCR product only has a DNA band, therefore directly the PCR product is carried out the TA clone, the test kit that uses is precious biological as 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, mixing is placed on 16 ℃ of reaction overnight.Adopting the heat shock conversion method will connect product changes in the bacillus coli DH 5 alpha.Use contains the LB solid medium screening positive clone of penbritin (ampicillin, Amp), selects several single bacterium colonies, shakes behind the bacterium with amplification
LrbZIP1Special primer identify multiple clone site and insert
LrbZIP1The clone, the clone who identifies is checked order final obtain
LrbZIP1Full-length cDNA is 979bp, analyzes by NCBI ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html) and finds that it comprises the opening code-reading frame (seeing sequence table) of a 429bp,
LrbZIP1Encoding one contains 142 amino acid whose Protein L rbZIP1, and its molecular weight is about 16.17 KDa, and iso-electric point is about 9.72, contain 1 cysteine residues (C), be positioned at the 129th, so monomeric protein do not have disulfide linkage to form, analyze by bioinformatics software SignalP 4.1
LrbZIP1Protein sequence detects it and whether has N end signal peptide.The result is presented at the existence that does not detect signal peptide among the LrbZIP1, shows that LrbZIP1 is a kind of non-secretory protein.
Embodiment 2: plant overexpression vector makes up
Adopt in a small amount extraction agent box (worker is given birth in Shanghai) the extraction insertion of SanPrep pillar plasmid DNA
LrbZIP1Escherichia coli plasmid pMD-18T-
LrbZIP1And the plasmid of plant expression vector pCAMBIA2300s, get 1 μ L and be used for integrity and the concentration height of agarose gel electrophoresis to detect the plasmid that extracted; Use restriction enzyme
EcoRI (TaKaRa) and
BamHI(TaKaRa) respectively to plasmid pMD-18T-
LrbZIP1Carry out double digestion (100 μ L system) with pCAMBIA2300s, reaction system and operating process are: get 20 μ L pMD-18T-
LrbZIP1With the 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 behind the mixing, place 37 ℃ of reaction overnight; All enzymes are cut the product point in sepharose, carry out electrophoresis, then right
LrbZIP1Fragment and pCAMBIA2300s carrier large fragment are carried out respectively glue and are reclaimed, and whole process uses SanPrep pillar DNA glue to reclaim test kit (worker is given birth in Shanghai); Get 1 μ L and reclaim product detects the recovery fragment by agarose gel electrophoresis size and concentration, place-20 ℃ to save backup.
Utilize T4 DNA Ligase(TaKaRa), with what reclaim
LrbZIP1Dna fragmentation and pCAMBIA2300s carrier segments couple together, and reaction system (20 μ L) and operating process are: get 10 μ L
LrbZIP1Dna 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 behind the mixing, 16 ℃ of water-bath reaction overnight then.Then adopt the heat shock conversion method will connect product and change in the bacillus coli DH 5 alpha, with the solid medium screening positive clone that contains 50mg/L kantlex (kanamycin, Km).Select single bacterium colony and shake bacterium, use amplification take bacterium liquid as template
LrbZIP1Special primer carry out PCR, pick out
LrbZIP1Clone with pCAMBIA2300s successfully is connected if the bacterial strain that detects is positive, adds glycerine and places-80 ℃ to save backup.
Adopt the pCAMBIA2300s-in (worker is given birth in the Shanghai) extraction of SanPrep pillar plasmid extraction test kit and the above-mentioned intestinal bacteria of purifying
LrbZIP1Plasmid.Use subsequently the frozen-thawed method with the plant expression vector pCAMBIA2300s-of above-mentioned structure
LrbZIP1Change in the agrobacterium tumefaciens lba4404 competent cell.Operation steps is: get 2 μ g pCAMBIA2300s-
LrbZIP1Plasmid adds and contains in the centrifuge tube of 200 μ L competent cells, ice bath 5 min behind the mixing change freezing 1min in the liquid nitrogen subsequently over to gently, then place rapidly 37 ℃ of water-bath 5 min, ice bath 2 min immediately add 800 μ L LB liquid culture based on 28 ℃ of shaking culture 4 h afterwards.Agrobacterium after the activation is applied on the LB solid medium that contains 50 mg/L Km 28 ℃ of static cultivations.Select single bacterium colony and shake bacterium, again with amplification
LrbZIP1Auele Specific Primer carry out PCR, detect pCAMBIA2300s-
LrbZIP1Whether change in the Agrobacterium, for positive colony, adding glycerine is placed on-80 ℃ and saves backup.
Embodiment 3: agriculture bacillus mediated Genetic Transformation in Higher Plants and transgenic plant screening
The transgene receptor of this experiment is tobacco, with tobacco seed with 75% alcohol-pickled 30 s, with after the sterilized water washing with the HgCl of 0.1 %
2Soak 8 min, and then wash several times with sterilized water, be seeded on the 1/2 MS substratum, 28 ℃ of dark 6 d that cultivate go to illumination box (25 ℃, 16h/d illumination) after the germination, use per month later on MS substratum subculture once.
From-80 ℃ of refrigerators, take out the pCAMBIA2300s-that contains that preserves
LrbZIP1The 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 the substratum muddiness.Draw the bacterium liquid of 1 mL muddiness to the LB solid medium that contains 50mg/L Km, cultivate 48 h for 28 ℃; Subsequently the Agrobacterium on the 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 2-3 h are with the activation Agrobacterium.
Get tobacco aseptic seedling leaf and be cut into 1 cm
2About the leaf dish, be soaked in above-mentioned containing in the MGL liquid nutrient medium that activates Agrobacterium fully, immerged time is 15 min, blot the bacterium liquid of blade surface with aseptic filter paper, the leaf dish placed carry out incubated at room temperature on the common substratum, 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 under the optical condition 2 days.
Leaf dish after the common cultivation forwarded to be added with seedling differentiation in the antibiotic MS screening culture medium, simultaneously the screening transgenic plant.The tobacco screening culture medium is MS+0.5 mg/L 6-BA+0.1 mg/L NAA+30 g/L sucrose+6 g/L agar+50 mg/L Km+200 mg/L cephamycins (cefotaxime sodium salt, Cef); During screening and culturing culturing bottle is transferred to illumination box and cultivates (25 ℃, 16h/d illumination, 8h/d is dark), after growing bud, tobacco uses 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, the tobacco regrowth is moved on the MS substratum that contains 50 mg/L Km it is taken root, select at last the regrowth of taking root preferably to do further detection.
Adopt the CTAB method to extract the genomic dna of transgenic tobacco plant blade, the genomic dna that extracts is got 1 μ L detect its integrity and concentration by agarose gel electrophoresis, use as template take the genomic dna of transfer-gen plant and increase
LrbZIP1Special primer carry out PCR, after PCR finishes, get 8 μ L products and be used for agarose gel electrophoresis detecting positive transfer-gen plant, the amplification of Partial Tobacco transfer-gen plant as shown in Figure 1,
LrbZIP1Transgene tobacco screens the positive transfer-gen plant of 42 strains altogether.
Embodiment 4: in the transgene tobacco
LrbZIP1Expression analysis and transfer-gen plant anti-mycotic activity analyze
The tender leaf of getting positive transgenosis individual plant and non-transgenic tobacco (wild-type) extracts total RNA, and reverse transcription generates cDNA the first chain, and as the template amplification
LrbZIP1Special primer carry out PCR, in each transgenosis individual plant of PCR interpretation of result
LrbZIP1The expression of transcriptional level, total RNA extract and the method for RT-PCR and embodiment 1 in identical, after PCR finishes, get 5 μ L for agarose gel electrophoresis, the detected result of part individual plant detects in 28 transgenosis individual plants as shown in Figure 2 altogether
LrbZIP1At transcriptional level expression is arranged, these individual plants be numbered 1～28.
Several fungies that the 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, treat to add when colony growth to diameter is about 2 ~ 3cm albumen, analyze the transfer-gen plant extracorporeal antifungal activity, have 5 kinds for the examination fungi: grape seat chamber bacterium (
Botrosphaeria dothidea), Fusarium oxysporum (
Fusarium oxysporum), Phomopsis (
PhomopsisSp.) fungi, Alternariaspp (
AlternariaSp.), Botrytis cinerea (
Botrytis cinerea).
For the albumen that prevents that other living contaminants from extracting, whole vegetable-protein leaching process all is aseptic techniques, at first get 1 g transgene tobacco individual plant (numbering is respectively 1,5,7,10,12) and wild-type blade and put into mortar, add 1 mL protein extract (1M NaCl, 0.1M sodium acetate, 1% PVP, pH6), fully grind; Change in the 1.5 mL centrifuge tubes 4 ℃ of standing over night behind the mixing, 4 ℃ of centrifugal 30 min(12,000 g/min over to), get supernatant in 1.5 new mL centrifuge tubes, and get an amount of 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, the total protein of parallel interpolation wild-type tobacco of while and blank (extracting the used solution of albumen), 28 ℃ cultivate several days afterwards observation respectively process the situation of fungal growth, and estimate accordingly
LrbZIP1The extracorporeal antifungal activity of transgene tobacco, the result as shown in Figure 3,
LrbZIP1Transgene tobacco albumen has very strong restraining effect to the growth of grape seat chamber bacterium, Phomopsis fungi, Fusarium oxysporum, and Alternariaspp is also had obvious inhibition activity.
Sequence table (SEQ ID)
＜110〉Kunming University of Science and Technology
＜120〉a kind of lilium regale wilson bZIP transcription factor gene
LrbZIP1And use
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 979
<212> DNA
<213>
Lilium regaleWilson
<400> 1
acatggggct tcccctcttc cccaccaatt ccagatctag ggtttccgag tttcccatct 60
ccagatctag ggtttccaag cctctcaatt tcagatctag ggtttccaag cttcagcttg 120
taatcctcat tcatgagctt cgtctcgttt tgatgagctt cgcggccttg agcttctgtt 180
tcctccactc cttttccgtc gtccttctct actggttcta tgccgtctct tgagccgatc 240
ttatactcat cacactcaat ttaactctta tagtcatagt tgttatctcg atcgctatct 300
cgaagatgtc tgcaaagacg gctagtcagt cgtccggttc ctccgccgga gccgccccca 360
ccgatgagcg gaagcgcaag aggatgctgt ccaaccgcga gtcggcgcgg cggtcccggg 420
tgaagaagca gcggcatctg gacgagctga tcaagcaggc ggcggagctg agggaggaga 480
atgctcggat cgtggctcgg acggatcagt tgactgcgcg gtacctgttg gtggagccgg 540
agaaccggtt cctgacggcg caagtggcgg agttgaccgc gaggctgcag tcgatgaact 600
cggtgttgcg gtttgtcaag gagtttagcg ggatggagat ggatattccg gatgttccgg 660
acccgcttat gaagcagtgg cagtttctct gcccggtgca gccgatcatg gcctctgcag 720
atatgttcca gtgatctagt ttgagtttca tgttattttg tgttactgaa ttgttggtac 780
tggttgtgtt tatatgtagt gttgtttgtt ggatctgagt actaaatcta tgttttttgc 840
tgaaactaat tattatgttc aattgattgt atgtttgatt aattggtttg tgtggcaacc 900
aaagggaatg tgtaacatca ttttgattct ttattgttac ctgtcttggc caaaaaaaaa 960
aaaaaaaaaa aaaaaaaaa 979
<210> 2
<211> 142
<212> PRT
<213>
Lilium regaleWilson
<400> 2
Met Ser Ala Lys Thr Ala Ser Gln Ser Ser Gly Ser Ser Ala Gly Ala
1 5 10 15
Ala Pro Thr Asp Glu Arg Lys Arg Lys Arg Met Leu Ser Asn Arg Glu
20 25 30
Ser Ala Arg Arg Ser Arg Val Lys Lys Gln Arg His Leu Asp Glu Leu
35 40 45
Ile Lys Gln Ala Ala Glu Leu Arg Glu Glu Asn Ala Arg Ile Val Ala
50 55 60
Arg Thr Asp Gln Leu Thr Ala Arg Tyr Leu Leu Val Glu Pro Glu Asn
65 70 75 80
Arg Phe Leu Thr Ala Gln Val Ala Glu Leu Thr Ala Arg Leu Gln Ser
85 90 95
Met Asn Ser Val Leu Arg Phe Val Lys Glu Phe Ser Gly Met Glu Met
100 105 110
Asp Ile Pro Asp Val Pro Asp Pro Leu Met Lys Gln Trp Gln Phe Leu
115 120 125
Cys Pro Val Gln Pro Ile Met Ala Ser Ala Asp Met Phe Gln
130 135 140
<210> 3
<211> 30
<212> DNA
＜213〉artificial sequence
<400> 3
ggatcccgtc cttctctact ggttctatgc 30
<210> 4
<211> 31
<212> DNA
＜213〉artificial sequence
<400> 4
gaattccatg aaactcaaac tagatcactg g 31
Claims (4)
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152465A (en) * | 2014-08-13 | 2014-11-19 | 昆明理工大学 | Lilium regale cytochrome b5 gene LrCyt-b5 and application thereof |
| CN107267526A (en) * | 2017-07-05 | 2017-10-20 | 昆明理工大学 | Pseudo-ginseng myb transcription factor gene PnMYB2 and its application |
| CN110747202A (en) * | 2019-11-13 | 2020-02-04 | 昆明理工大学 | Lilium regale WRKY transcription factor gene LrWRKY11 and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1472223A (en) * | 2002-07-30 | 2004-02-04 | 中国农业科学院生物技术研究所 | A maize bZIP-like transcription factor and its coding gene and application |
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2013
- 2013-06-26 CN CN201310258369.8A patent/CN103320448B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1472223A (en) * | 2002-07-30 | 2004-02-04 | 中国农业科学院生物技术研究所 | A maize bZIP-like transcription factor and its coding gene and application |
Non-Patent Citations (2)
| Title |
|---|
| RAO J. ET AL.: "JZ390956", 《EMBL-EBI》 * |
| 杨颖 等: "植物bZIP转录因子的研究进展", 《麦类植物学报》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104152465A (en) * | 2014-08-13 | 2014-11-19 | 昆明理工大学 | Lilium regale cytochrome b5 gene LrCyt-b5 and application thereof |
| CN107267526A (en) * | 2017-07-05 | 2017-10-20 | 昆明理工大学 | Pseudo-ginseng myb transcription factor gene PnMYB2 and its application |
| CN107267526B (en) * | 2017-07-05 | 2019-07-16 | 昆明理工大学 | Panax notoginseng MYB transcription factor gene PnMYB2 and its application |
| CN110747202A (en) * | 2019-11-13 | 2020-02-04 | 昆明理工大学 | Lilium regale WRKY transcription factor gene LrWRKY11 and application thereof |
| CN110747202B (en) * | 2019-11-13 | 2021-09-14 | 昆明理工大学 | Lilium regale WRKY transcription factor gene LrWRKY11 and application thereof |
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| CN103320448B (en) | 2014-09-03 |
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