CN108018294B - Panax notoginseng plant hormone binding protein genePnPhBP1And applications - Google Patents

Abstract

The invention discloses a pseudo-ginseng plant hormone binding protein genePnPhBP1The nucleotide sequence is shown as SEQ ID NO: 1, the coding sequence is shown as SEQ ID NO: 2, the invention is proved by related technical research of functional genomicsPnPhBP1The gene has the function of improving the plant antifungal property, and the invention is used for resisting the fungiPnPhBP1The gene is constructed on a plant expression vector and is transferred into tobacco for over-expression, and as a result, the transgenic tobacco plant has strong in-vitro antifungal activity, and the experimental result shows that the over-expression is realizedPnPhBP1The transgenic tobacco has obvious inhibiting effect on the growth of three fungi, i.e. colletotrichum gloeosporioides, botrytis cinerea, fusarium solani and the like.

Description

Panax notoginseng plant hormone binding protein genePnPhBP1And applications

Technical Field

The invention relates to the field of molecular biology and genetic engineering related technology research, in particular to a panax notoginseng plant hormone binding protein genePnPhBP1And application thereof.

Background

Plant diseases are a very troublesome problem in agricultural production, especially fungal diseases, accounting for about 80% of the total plant diseases, and seriously affecting the yield and quality of crops. The traditional disease control method achieves certain effect, firstly, resistant varieties are cultivated by the traditional breeding method, secondly, chemical pesticides are used, and thirdly, cultivation systems such as crop rotation are adopted. However, these methods have disadvantages, such as long cultivation period of resistant varieties, high chemical pesticide residue, and easy environmental pollution, and time and labor consuming cultivation system adjustment, so that the conventional methods for controlling plant diseases can not completely solve the problems. With the establishment and development of recombinant DNA technology, the gene engineering technology is utilized to cultivate new varieties of disease-resistant plants, which has achieved initial effect and is expected to solve the problem of fungal diseases fundamentally.

A series of physiological, biochemical and molecular biological reactions occur in plants through disease Resistance gene-mediated defense processes, which are reactions that initiate from a pathogenic infection point (Hypersensitive Response, HR) and extend to Systemic or Acquired Resistance (SAR) in distant tissues, under the control of a signaling network. Plant hormones (phytohormons) widely participate in signal systems of disease-resistant defense reactions, such as salicylic acid, jasmonic acid, ethylene and the like, and finally induce the expression and metabolic changes of a series of defense reaction genes to generate resistance by regulating the expression and signal amplification of key genes. Plant hormones are chemical messengers that coordinate many cellular functions, including (but not limited to) ten major classes: auxins, cytokinins, gibberellins, abscisic acid, brassinosteroids, ethylene, jasmonates, polypeptide hormones, salicylic acid and strigolactones (Santner A, Calderon-Villalobos L I, Estelle M. Plant hormons area versatille chemical regulators of Plant growth. Nat Chem biol. 2009, 5: 301. 307). A variety of plant hormone-binding proteins (phbps) also play an important role in the plant hormone-mediated signaling network.

Gibberellins (e.g., gibberellic acid) are diterpene tetracyclic or pentacyclic growth regulators that induce seed development, germination, organ elongation, and flowering (Yamaguchi S. Gibberellin metabolism and its regulation. Annu Rev Plant biol. 2008, 59: 225-. Gibberellins were first introduced in gibberellins (A), (B), (C)Gibberella fujikuroi) It is found that a fungal pathogen of rice, causes extreme elongation of the stem and ultimately leads to plant blight and death. Plants produce endogenous gibberellins, the intracellular levels of which are regulated by a negative feedback loop, and in addition, the concentrations of auxin and ethylene also regulate the intracellular levels of gibberellins (fly C M, Sun T P. A DELLAcate balance: the roll of gibberella in Plant morphology. Curr Opin Plant biol. 2005, 8: 77-85; Yamaguchi S. gibberella metabolism and its regulation. Annu Rev Plant biol. 2008, 59: 225-. The gibberellin receptor is called gibberellin-insensitive dwarf 1 protein (GID 1),gid1loss of function mutations in the genes result in plant dwarfing (Peng J, Richards D E, Hartley N M, Murphy G P, Devos K M, Flintham J E, Beales J, Fish L J, Worland A J, Pelia F, Sudhakar D, Christou P, Snape J W, Gale M D, Harberd N P. 'Green recovery' genes amino mutant responses models, Nature 1999, 400: 256-261). Murase et al Arabidopsis thaliana (Arabidopsis thaliana) The complex of GID1 with Gibberellin was structurally studied (Murase K, Hirano Y, Sun T P, Hakoshima T. Gibberellin-induced DELLA registration by the gibberella receiver GID1. Nature 2008, 456: 459-463). The GID1 receptor can bind to DELLA proteins with conserved Asp-Glu-Leu-Leu-Ala N-terminal sequences, which are negative feedback regulators of gibberellin response (Schwechheimer C. inversion binding gibberic acid signaling- -are we heat layer et. Curr Opin Plant biol. 2008, 11: 9-15; Schwechheimer C, Willive B C. shortening light on gibberic acid signaling. Curr Opin Plant biol. 2009, 12: 57-62). Gibberellin binding to GID1 initiates the formation of GID1-DELLA complex, and DELLA proteins can no longer act as transcriptional repressors of gibberellin-dependent genes, but are ubiquitinated and degraded.

The disease course-associated protein 10 (pathologenesis related protein 10) is very small (only 19 kDa at the most) and is usually a monomeric, slightly acidic cytoplasmic plant-specific protein (Fernandes H, Michalska K, Sikorski M, Jaskolski M. Structural and functional assays of PR-10 proteins FEBS J. 2013, 280: 1169-. The structural conservation of the PR10 protein is well established, and the PR10 protein has a typical folding property. The most prominent feature of the PR10 fold is the formation of a large hydrophobic cavity between the helices α 3 and β -sheet, apparently the binding site for the PR-10 ligand. Crystal studies confirmed the possibility of binding of the PR10 protein to cytokinins (Fernandes H, Bujacz A, Bujacz G, Jelen F, Jasinski M, Kachlick P, Otlewski J, Sikorski M, Jaskolski M, cytokine-induced structural adaptation of a Lupinus luteus PR-10 protein, FEBS J2009, 276: 1596-. PR10, which has a very low sequence similarity, was identified as a subfamily of Cytokinin-Specific Binding Proteins (CSBP), and in addition, two CSBP MtCSBP and VrCSBP were shown to bind gibberellin as well (Ruszkowski M, Sliweak J, Cieseilska A, Barciszewski JSikorski M, Jaskolski M, Specific binding of gibberellac acid by cytokine-Specific binding proteins, a new aspect of plant hormone-binding proteins with the PR-10 fold, Acta Crystalographica, 2014, 70(Pt 7): 2032-. The above studies indicate that PR10 does not specifically bind only to cytokinins, and Ruszkowski et al suggested the substitution of PhBP for CSBP as a term.

Notoginseng [ radix ], [ sic ]Panax notoginseng (Burk) F.H. Chen]Is prepared from Panax of Araliaceae (Araliaceae)Panax) Herbaceous plants, also known as pseudo-ginseng and jinbuhui, are important traditional and rare Chinese medicinal materials in China, are applied to minority nationalities (Zhuang, Miao, Yao and Yi nationality) in southwest areas of China at first and then gradually introduced into the central areas along with communication among the nationalities and propagation of military travel and merchant. The active ingredients in the panax notoginseng are mainly saponins, so that the panax notoginseng has the effects of relieving swelling and pain, resisting inflammation and aging, regulating the immunologic function of a human body, eliminating fatigue and delaying aging, and the panax notoginseng saponins can also effectively improve the memory, so that the panax notoginseng saponins are long-history natural resources used as both medicine and food. Although the demand for pseudo-ginseng medicinal materials rises year by year, the yield of pseudo-ginseng is not greatly increased, and the long growth period and multiple diseases of pseudo-ginseng are one of the main reasons. The disease resistance genetic breeding research of the panax notoginseng is weak in foundation and becomes a limiting factor for the healthy development of the panax notoginseng industry. The deep research on the disease-resistant molecular mechanism of the panax notoginseng, especially the disclosure of the signal transduction of disease-resistant defense reaction and the discovery of disease-resistant functional genes, is helpful for the stable promotion of the panax notoginseng disease-resistant genetic breeding work.

Disclosure of Invention

The invention aims to provide a full-length gene for coding a plant hormone binding protein cloned from pseudo-ginsengPnPhBP1Plant hormone binding protein genePnPhBP1The nucleotide sequence is shown as SEQ ID NO: 1, the gene cDNA full-length sequence is 623bp, comprises a 465bp open reading frame, a 56bp 5 'untranslated region and a 102bp 3' untranslated region, and codes are shown as SEQ ID NO: 2 in the sequence table 2.

In the inventionPnPhBP1The coding region of the gene is shown in a sequence table SEQ ID NO: 1 from 57 to 521And (4) columns.

The present invention separates and clones complete cDNA segment of antifungal related gene of notoginseng and utilizes Agrobacterium tumefaciens (A. tumefaciens)Agrobacterium tumefaciens) The target gene is transferred into a receptor plant and is overexpressed, whether the gene has antifungal capacity is verified through further experiments, a foundation is laid for improving the capacity of tobacco and other plants for resisting fungal diseases by utilizing the gene in the later period, and the inventor names the gene asPnPhBP1。

The invention relates to the separation ofPnPhBP1And identifying the functions of the DNA fragment, wherein the DNA fragment is shown in a sequence table SEQ ID NO: 1, the sequence analysis of the gene is carried out, and the discoveryPnPhBP1The full-length cDNA is 623bp, and comprises a 465bp Open Reading Frame (ORF), a 56bp 5 'untranslated region (UTR), and a 102bp 3' UTR, wherein the ORF encodes a protein with 154 amino acids.PnPhBP1The encoded protein has the conserved motif glycine-rich loop of PR10 protein, and the BLASTp search result shows that PnPHBP1 and carrot (A), (B) and (C)Daucus carota) Peach (a), (b)Prunus persica) Macleaya cordata (Macleaya cordata) And castor (Ricinus communis) The similarities of (a) are 76%, 66%, 62% and 61%, respectively, indicating that it belongs to the plant hormone binding protein in notoginseng. The overexpression sequence table SEQ ID NO: 1 can enhance the resistance of tobacco to botrytis cinerea: (Botrytis cinerea) Fusarium solani (F.solani) (II)Fusarium solani) Colletotrichum gloeosporioides (B) ((B))Colletotrichum gloeosporioides) Resistance of (2).

The invention relates to a pseudo-ginseng plant hormone binding protein genePnPhBP1The method is applied to improving the resistance of tobacco to botrytis cinerea, fusarium solani and colletotrichum gloeosporioides, and comprises the following specific operations:

(1) using amplificationPnPhBP1The specific primer is used for extracting total RNA from pseudo-ginseng roots and amplifying the total RNA by reverse transcription-polymerase chain reaction (RT-PCR)PnPhBP1Then connecting the coding region to a pMD-18T vector, and obtaining a clone with a target gene through sequencing;

(2) by using restriction endonucleasesEnzymePstI andEcoRi enzyme digestion of pMD-18T-PnPhBP1Recovering the carrier by glue to obtain target gene segment, using the same endonuclease to enzyme-cut plant expression carrier pCAMBIA2300s, recovering the glue to obtain the required carrier large segment, and recovering the obtained carrier large segmentPnPhBP1Connecting the gene fragment with the pCAMBIA2300s fragment to construct a plant overexpression vector, and then transferring the constructed recombinant vector into tobacco to express through the mediation of agrobacterium tumefaciens;

(3) screening transformants by using a resistance marker on the recombinant vector T-DNA, obtaining a real transgenic plant through PCR and RT-PCR detection, analyzing the inhibition activity of the transgenic plant protein on the growth of fungi, and finally screening the transgenic plant with obviously enhanced resistance to the fungi.

The invention provides a new method for improving the resistance of plants to fungal diseases, the defects of traditional breeding can be overcome by cultivating disease-resistant plants by means of genetic engineering, the breeding period is shortened, the operation is simple, and high-resistance materials are easy to obtain. The invention is derived from notoginsengPnPhBP1The gene can enhance the resistance of plants to fungi, and can be introduced into tobacco to produce new varieties and new materials with fungal resistance. The cultivation of resistant plant varieties and materials by using genetic engineering technology has obvious advantages and irreplaceable importance. The invention not only can provide convenience for large-scale production of crops, flowers and the like, greatly reduces the use of chemical pesticides, but also can save the cost for agricultural production and reduce the environmental pollution, thereby having wide market application prospect.

Drawings

FIG. 1 is a drawing of the present inventionPnPhBP1Schematic diagram of PCR detection result of transgenic tobacco genome DNA, in which: the Marker is DL2000 DNA Marker (Dalibao biology); the positive control is plasmid pMD-18T-PnPhBP1PCR products as templates; WT is the product of PCR using total DNA of non-transgenic tobacco (wild type) as template;

FIG. 2 shows the positivity of the present inventionPnPhBP1In transgenic tobaccoPnPhBP1A graph of the results of expression analysis at the transcriptional level; in the figure: marker is DL2000 DNA Marker (Dalibao biology); WT is non-transgenic tobacco total RNA reverse transcription cDNA as templatePCR products; the positive control was plasmid pMD-18T-PnPhBP1A PCR product as a template;

FIG. 3 is a drawing of the present inventionPnPhBP1The in vitro bacteriostatic activity effect graph of the transgenic tobacco; in the figure, a, b and c are respectively botrytis cinerea, colletotrichum gloeosporioides and fusarium solani; WT is the total protein of wild type tobacco; buffer is a blank control, i.e. a no protein control (Buffer used for protein extraction).

Detailed Description

The present invention is further illustrated by the following figures and examples, but the scope of the present invention is not limited to the above description, and the examples are conventional methods unless otherwise specified, and reagents used are conventional commercially available reagents or reagents formulated according to conventional methods unless otherwise specified.

Example 1:PnPhBP1full-Length Gene cloning and sequence analysis

Inoculating fusarium solani to pseudo-ginseng, extracting total RNA from roots inoculated for 24 hours, grinding the inoculated pseudo-ginseng roots into powder by using liquid nitrogen, transferring the powder into a centrifuge tube, extracting the total RNA by using a guanidinium isothiocyanate method, synthesizing a first cDNA chain by using reverse transcriptase M-MLV (promega) and using the total RNA as a template, wherein a reaction system and an operation process are as follows: taking 5 μ g of total RNA, adding 50 ng oligo (dT), 2 μ L dNTP (2.5 mM each) and DEPC water in turn to make the reaction volume be 14.5 μ L; after mixing, heating and denaturation at 70 ℃ for 5 min, then rapidly cooling on ice for 5 min, then adding 4 μ L of 5 XFirst-stand buffer, 0.5 μ L of RNase (200U) and 1 μ L M-MLV (200U) in sequence, mixing and centrifuging for a short time, bathing at 42 ℃ for 1.5 h, taking out, heating at 70 ℃ for 10 min, and terminating the reaction. The first strand cDNA is synthesized and stored at-20 deg.C for further use.

Amplifying target gene using synthesized first strand cDNA as templatePnPhBP1The sequences of the upstream primer and the downstream primer are respectively

And

. Advantage is taken^TM2 PCR Enzyme (Clontech) amplificationIncreasing a target gene; and (3) PCR reaction conditions: 1 min at 95 ℃; 30 cycles of 94 ℃ for 30s, 61 ℃ for 30s, 72 ℃ for 40 s; 5 min at 72 ℃; the reaction system (10. mu.L) was 1. mu.L of cDNA, 1. mu.L of 10 × Advantage 2 PCR Buffer, 0.5. mu.L of 50 × dNTP Mix (10 mM each), 0.2. mu.L of forward primer (10. mu.M), 0.2. mu.L of reverse primer (10. mu.M), 0.2. mu.L of Advantage 2 PCR Polymerase Mix, 6.9. mu.L of PCR-Grade water; after the PCR was completed, 5. mu.L of the resulting mixture was subjected to agarose gel electrophoresis to examine the specificity and size of the amplified product.

The obtained PCR product only has one DNA band, so TA cloning is directly carried out on the PCR product, the used kit is pMD18-T vector kit (Dalianbao biology), and the reaction system and the operation process are as follows: mu.L of the PCR product was taken, and 1. mu.L of pMD18-T vector (50 ng/. mu.L) and 2.5. mu.L of 2 × Ligation solution I were added in this order, mixed well and allowed to react overnight at 16 ℃. The ligation product was transformed into E.coli DH 5. alpha. using a heat shock transformation method. Screening positive clones with LB solid medium containing ampicillin (Ampicillin, Amp), selecting several single colonies, shaking, and amplifyingPnPhBP1Identifying the multiple cloning site insertionPnPhBP1The clones identified are sequenced and finally obtainedPnPhBP1The full-length cDNA was 623bp, which was found to contain a 465bp open reading frame by NCBI ORF finder (http:// www.ncbi.nlm.nih.gov/gorf. html) analysis (see sequence listing),PnPhBP1encodes a protein PnPhBP1 containing 154 amino acids, has a molecular weight of about 16.99 KDa, an isoelectric point of about 5.08, and contains 1 cysteine residue. Analysis by means of bioinformatics software SignalP 4.1PnPhBP1The encoded protein sequence, and detecting whether the protein sequence has an N-terminal signal peptide. The results are shown inPnPhBP1The presence of the signal peptide was not detected, indicating that PnPhBP1 is a non-secreted protein.

Example 2: construction of plant overexpression vectors

The insertion is extracted by adopting a SanPrep column type plasmid DNA small extraction kit (Shanghai worker)PnPhBP1The Escherichia coli plasmid pMD-18T-PnPhBP1And the plasmid of the plant expression vector pCAMBIA2300s, and 1 μ L of the plasmid was subjected to agarose gel electrophoresis to detectThe integrity and concentration of the extracted plasmid are high and low; using restriction endonucleasesPstI (TaKaRa) andEcoRi (TaKaRa) plasmid pMD-18T-PnPhBP1And pCAMBIA2300s (100 mu L system), wherein the reaction system and the operation process are as follows: taking 20. mu.L of pMD-18T-PnPhBP1And pCAMBIA2300s plasmid, 10. mu.L 10 XK buffer, and 4. mu.LPstI、6 μLEcoRI、60 μL ddH₂O, mixing uniformly, centrifuging for a short time, and reacting at 37 ℃ overnight; all the products of the digestion are spotted in agarose gel for electrophoresis, and thenPnPhBP1Respectively carrying out gel recovery on the fragment and the large fragment of the pCAMBIA2300s vector; taking 1 microliter of the recovered product, detecting the size and concentration of the recovered fragment by agarose gel electrophoresis, and storing at-20 ℃ for later use.

The recovered DNA was purified by using T4 DNA Ligase (TaKaRa)PnPhBP1The DNA fragment and the pCAMBIA2300s vector fragment were ligated, and the reaction system (20. mu.L) and the procedure were as follows: taking 10 μ LPnPhBP1The DNA fragment was sequentially added with 2. mu.L of pCAMBIA2300s vector DNA, 2. mu.L of 10 XT 4 DNA Ligase Buffer, 1. mu. L T4 DNA Ligase, and 5. mu.L of ddH₂And O, mixing uniformly, centrifuging for a short time, and then carrying out water bath at 16 ℃ for overnight reaction. The ligation product was then transferred into E.coli DH 5. alpha. by heat shock transformation, and positive clones were selected on a solid medium containing 50 mg/L kanamycin (Km). Selecting single colony shake bacteria, taking bacteria liquid as template for amplificationPnPhBP1The specific primers of (1) are subjected to PCR, and selectedPnPhBP1If the detected strain is positive, the clone successfully connected with pCAMBIA2300s is added with glycerol and stored at-80 ℃ for later use.

Extraction and purification of pCAMBIA2300s-PnPhBP1Plasmid, then freezing and thawing the plant expression vector pCAMBIA2300s-PnPhBP1Transferred into Agrobacterium tumefaciens LBA4404 competent cells. The operation steps are as follows: taking 2 μ g of pCAMBIA2300s-PnPhBP1The plasmid is added into a centrifuge tube containing 200 mu L of competent cells, the mixture is gently mixed and then is subjected to ice bath for 5 min, then the mixture is transferred into liquid nitrogen to be frozen for 1 min, then the mixture is rapidly placed in a water bath at 37 ℃ for 5 min, then is subjected to ice bath for 2 min immediately, and is added with 800 mu L of LB liquid culture medium to be subjected to shaking culture at 28 ℃ for 4 h. After activation, the activatedAgrobacterium was spread on LB solid medium containing 50 mg/L Km and cultured at 28 ℃ in a static state. Selecting single colony shake bacteria, and amplifyingPnPhBP1The specific primer of (2) is used for PCR to detect pCAMBIA2300s-PnPhBP1If the positive clone is transferred into agrobacterium, adding glycerol into the positive clone, and storing the positive clone at-80 ℃ for later use.

Example 3: agrobacterium-mediated genetic transformation of plants and transgenic plant screens

The transgenic recipient in this experiment was tobacco, tobacco seeds were soaked in 75% ethanol for 30s, washed with sterile water and then washed with 0.1% HgCl₂Soaking for 8 min, washing with sterile water for several times, sowing on 1/2MS culture medium, dark culturing at 28 deg.C for 6 d, germinating, transferring to light incubator (25 deg.C, 16h/d light), and subculturing with 1/2MS culture medium once a month.

The preserved liquid containing pCAMBIA2300s was taken out from the-80 ℃ refrigeratorPnPhBP1Agrobacterium LBA4404 strain of plasmid was inoculated into 5 mL LB liquid medium containing 50 mg/L Km and 20 mg/L rifampicin, and cultured at 28 ℃ until the medium became turbid. Sucking 1 mL of turbid bacterial liquid to an LB solid culture medium containing 50 mg/L Km, and culturing for 48 h at 28 ℃; then, appropriate amount of the agrobacteria on LB solid medium was scraped and inoculated into MGL liquid medium supplemented with 20 mg/L acetosyringone, and shake-cultured at 28 ℃ for 2-3 h to activate the agrobacteria.

Cutting leaves of aseptic seedling of tobacco into 1 cm²And completely soaking the left and right leaf discs in the MGL liquid culture medium containing the activated agrobacterium for 15 min, sucking bacterial liquid on the surfaces of the leaves by using sterile filter paper, placing the leaf discs on a co-culture medium for room temperature culture, wherein the co-culture medium for tobacco transformation is MS +0.02 mg/L6-BA +2.1 mg/L NAA +30 g/L sucrose +6 g/L agar, and co-culturing for 2 days at 22 ℃ in the absence of light.

Transferring the co-cultured leaf discs to an MS screening culture medium added with antibiotics to be divided into seedlings, and screening transgenic plants. The tobacco screening culture medium is MS +0.5 mg/L6-BA +0.1 mg/L NAA +30 g/L sucrose +6 g/L agar +50 mg/L Km +200 mg/L cephamycin (cefixime sodium salt, Cef); during screening culture, the culture bottle is transferred to an illumination culture box for culture (25 ℃, 16h/d illumination and 8h/d darkness), after the tobacco grows out of buds, the MS culture medium containing 50 mg/L Km and 200 mg/L Cef is used for subculture, the regeneration plant needs to be further screened because the callus differentiation rate of the tobacco is higher, the tobacco regeneration seedling is transferred to the MS culture medium containing 50 mg/L Km to root the tobacco regeneration seedling, and finally the regeneration seedling with better rooting is selected for further detection.

Extracting genome DNA of transgenic tobacco plant leaf by CTAB method, collecting 1 μ L of the extracted genome DNA, detecting its integrity and concentration by agarose gel electrophoresis, and amplifying with the genome DNA of transgenic plant as templatePnPhBP1After the PCR is finished, 8 mu L of the product is used for agarose gel electrophoresis to detect positive transgenic plants, the amplification result of part of tobacco transgenic plants is shown in figure 1,PnPhBP1and co-screening 53 positive transgenic plants from the transgenic tobacco.

Example 4: in transgenic tobaccoPnPhBP1Expression analysis and antifungal Activity analysis of transgenic plants

Taking positive transgenic single plant and tender leaf of non-transgenic tobacco (wild type) to extract total RNA, reverse transcribing to generate first strand cDNA, and using it as template to make amplificationPnPhBP1The specific primers are used for carrying out PCR, and each transgenic individual is analyzed according to the PCR resultPnPhBP1The expression of transcription level, total RNA extraction and RT-PCR were performed in the same manner as in example 1, after PCR was completed, 5. mu.L of the DNA was subjected to agarose gel electrophoresis, and the results of detection of some individuals were shown in FIG. 2, and 36 transgenic individuals were detected in totalPnPhBP1The expression was carried out at the transcriptional level in a large amount, and the numbers of these individuals were 1 to 36.

Inoculating a plurality of fungi stored in a laboratory on a PDA solid culture medium (200 g/L of potatoes, 15 g/L of agar and 20 g/L of glucose), carrying out dark culture at 28 ℃, adding protein when bacterial colonies grow to the diameter of about 2-3 cm, and analyzing the in-vitro antifungal activity of a transgenic plant. In order to prevent the extracted protein from being polluted by other mixed bacteria, the whole plant protein extraction process is aseptic operation, firstly, 1 g of single transgenic tobacco plant (numbered 7, 12, 28 and 35 respectively) and wild type leaves are put into a mortar, and 1 mL of protein extracting solution (1M NaCl, 0.1M) is addedSodium acetate, 1% PVP, pH 6), thoroughly ground; transferring into 1.5 mL centrifuge tube, mixing, standing overnight at 4 deg.C, centrifuging at 4 deg.C for 30 min (12,000 g/min), collecting supernatant, and determining total protein concentration with ultraviolet spectrophotometer. The total protein concentration of the transgenic and wild type plants is adjusted to 0.5 mug/muL, then 20 muL is respectively dropped on the sterile filter paper of each fungus culture medium, the total protein of different transgenic tobacco plants is added on the plate of each fungus, the total protein of the wild type tobacco and a blank control (solution for extracting the protein) are added in parallel, the growth of each treated fungus is observed after the fungus is cultured for several days at 28 ℃, and the growth of each treated fungus is evaluated according to the total protein concentrationPnPhBP1The results of the in vitro antifungal activity of the transgenic tobacco are shown in FIG. 3,PnPhBP1the transgenic tobacco protein has strong inhibiting effect on the growth of botrytis cinerea, fusarium solani and colletotrichum gloeosporioides.

Sequence listing

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Claims (2)

1. Panax notoginseng plant hormone binding protein genePnPhBP1The method is characterized in that: the nucleotide sequence is shown as SEQ ID NO: 1, the coding sequence is shown as SEQ ID NO: 2 in the sequence table 2.

2. The notoginseng plant hormone binding protein gene as set forth in claim 1PnPhBP1In increasing the resistance of tobacco to botrytis cinerea (Botrytis cinerea) Fusarium solani (F.solani) (II)Fusarium solani) Colletotrichum gloeosporioides (B) ((B))Colletotrichum gloeosporioides) Use in resistance.

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