WO2012079370A1 - 大丽轮枝菌蛋白质激发子基因、蛋白序列及其应用 - Google Patents

大丽轮枝菌蛋白质激发子基因、蛋白序列及其应用 Download PDF

Info

Publication number
WO2012079370A1
WO2012079370A1 PCT/CN2011/077486 CN2011077486W WO2012079370A1 WO 2012079370 A1 WO2012079370 A1 WO 2012079370A1 CN 2011077486 W CN2011077486 W CN 2011077486W WO 2012079370 A1 WO2012079370 A1 WO 2012079370A1
Authority
WO
WIPO (PCT)
Prior art keywords
protein
plant
tobacco
pevdl
polynucleotide
Prior art date
Application number
PCT/CN2011/077486
Other languages
English (en)
French (fr)
Inventor
邱德文
曾洪梅
杨秀芬
郭立华
袁京京
王炳楠
Original Assignee
中国农业科学院植物保护研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国农业科学院植物保护研究所 filed Critical 中国农业科学院植物保护研究所
Publication of WO2012079370A1 publication Critical patent/WO2012079370A1/zh

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance

Definitions

  • the present invention relates to a peptide having more than 20 amino acids, a nucleotide sequence encoding the same, and an application thereof, and particularly to a Verticillium from Verticillium which enhances plant resistance and induces plant defense responses.
  • Dahliae a protein and a nucleotide sequence encoding the same and its use. Background technique
  • Cotton Verticillium Wilt is a highly harmful vascular bundle disease. It brings great harm to China's cotton production every year, and seriously affects the quality and output of China's cotton production.
  • Verticillium dahliae is classified into the genus Hymenoptera, Trichosporum, Trichosporon, Pseudomonas, Verticillium, and belongs to the two species of Verticillium and Verticillium The most harmful species in China is Verticillium dahliae.
  • chemical control and breeding varieties are mainly used for the prevention and control of plant diseases. However, they often have the drawbacks that cannot be eliminated.
  • the protein produced by it is an important effector. It transmits the elicitor signal of the pathogen by recognizing and acting on the receptor molecule of the plant cell surface or subcellular component, initiating the plant's defense system, inducing the local tissue of the plant to produce allergic cell necrosis, and passing through the system of local cell signaling substances. Delivery causes the plant to eventually produce systemic acquired resistance.
  • the elicitors in the world's research on Verticillium dahliae mainly play a toxin role.
  • a number of active ingredients have been isolated from Verticillium dahliae that can cause plant disease resistance or cause plant pathogenesis.
  • Buchner, V., etc. isolated a lipopolysaccharide complex PLPC (protein-lipopolysaccharide complex) in the culture solution of Verticillium dahliae, which can induce pathogenic symptoms similar to Verticillium dahliae (Buchner). , V. et al., Physiol. Mol. Plant Path., 1989, 35: 253-269) ; at the same time, Kazakov, I., etc.
  • the protein can induce the outbreak of reactive oxygen species and the expression of related PR genes, and induce the synthesis of phytoalexins such as gossypol in cotton suspension cells at a lower concentration (APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2004, 70, 4989-4995; Patent No. ZL 200310108076.8).
  • An object of the present invention is to provide an isolated protein derived from Verticillium dahliae and a polynucleotide sequence encoding the same, which are capable of enhancing plant resistance and inducing a plant defense response, and uses thereof.
  • the isolated polynucleotide of the present invention wherein the polynucleotide sequence encoding the amino acid sequence as shown in SEQ ID NO: 2 is represented by SEQ ID NO: 1.
  • a vector comprising the above isolated polynucleotide.
  • a genetically engineered host cell comprising the above vector.
  • a protein preparation method of the present invention which comprises the following methods:
  • a protein having the amino acid sequence of SEQ ID NO: 2 is isolated from the culture.
  • the protein of the present invention refers to a protein derived from a culture solution of Verticillium dahliae, a supernatant of a culture solution, or a bacterial cell.
  • FIG. 1 shows the purified natural protein from Verticillium dahliae from Verticillium dahliae.
  • the amino acid sequence obtained by MALDI-TOF and de novo sequencing is compared with the amino acid sequence of the fusion expressed protein, and the underlined part is the amino acid sequence obtained by sequencing the natural protein, and the whole sequence is the amino acid sequence of the fusion expressed protein;
  • FIG. 2 is treated with water in the present invention. The leaves were irradiated with TMV 3 days later, and the tobacco leaves were photographed 3 days after inoculation;
  • Fig. 3 is a tobacco leaf which was treated with 1.0 ⁇ ⁇ , 50 w L His-PevDl for 3 days, and was inoculated with TMV 3 days after inoculation. detailed description
  • the Verticillium dahliae strain of Verticillium dahliae was streaked onto a PDA plate, cultured at 25 °C for 2 weeks, and the colony edge was picked and inoculated into a 500 ml flask containing 200 mL of liquid medium, wherein the liquid medium was Czapek- Dox was added with 1% hydrolyzed lysine (Sigma), and cultured at 25 ° C, 150 r / min for 14 days to obtain a fermentation broth of Verticillium dahliae.
  • the preparation method of the PDA plate is as follows: Take 200 g of peeled potato, cut into pieces, add 1000 ml of water and boil for 30 minutes, filter out the potato pieces, make up the filtrate to 1000 ml, add 20 g of glucose, 15 g of agar, wait for glucose and The agar was dissolved, dispensed, and sterilized at 121 °C for 30 minutes.
  • the preparation method of Czapek-Dox is: sodium nitrate 3.0g, anhydrous dibasic potassium phosphate 1.0g, magnesium sulfate heptahydrate 0.5g, potassium chloride KCl 0.5g, ferrous sulfate heptahydrate 0.01g, sucrose 30.0g (Beijing Chemical plant, analytical grade), pH 5.6, add deionized water to 1L.
  • Example 2 The fermentation broth obtained in Example 1 was centrifuged at 4 ° C, 5000 rpm for 1 h or at 4 ° C, 13,000 rpm for 30 min, and the supernatant was collected and filtered with a filter having a pore size of 0.45 mm (Whatman). Twice, until there are no cells. Ammonium sulfate powder was added to precipitate the target protein in the supernatant with a final concentration of ammonium sulfate of 80%, and dialyzed for 48 hours at 4 ° C. Finally, the obtained extracellular total protein was dissolved in Tris-HCl buffer (20 mM, pH 8.0). The final protein concentration was 100 ⁇ g/mL. The protein content was measured by a GF ATM protein assay kit ( Thermo Scientific) at a wavelength of 590 nm by a GF-M2000 microplate reader (Shandong Gaomi Rainbow Analytical Instruments Co., Ltd.).
  • Bioactivity assay Dilute the crude protein solution to 50 yg / mL with 2-7 grown leaves of tobacco with 5-7 true leaves. Use 50 mL of syringe without syringe to remove 50 ⁇ L of solution from the leaves. The back side was injected into the leaves, and Tris-HCl buffer and 50 ⁇ g/mL bovine serum albumin solution were used as controls. After 24 hours, it was observed whether or not necrotic spots were formed.
  • Example 3 Isolation, Purification and Biological Activity Determination of Protein Elicitors
  • the resulting total protein was further purified using an AKTA explore 10 (GE Healthcare) protein purifier.
  • the sample was first linearly eluted with NaCl using an HP Q HiTrapTM anion exchange column (GE Healthcare) (0%-100%, 30 min)
  • HP Q HiTrapTM anion exchange column GE Healthcare
  • the 6 protein elution peaks were obtained, and the protein elicitor activity of each of the obtained protein elution peaks was detected according to the biological activity detection method in Example 2, and the protein concentration in each eluted peak was adjusted to 50 ⁇ g/mL.
  • the third protein peak strongly induced an allergic reaction in tobacco; the protein peak was further purified by pheny HP hydrophobic chromatography column (GE Healthcare) and electroelution to obtain a single active protein.
  • the single protein peak was detected as a single band by 15% denaturing polyacrylamide gel electrophoresis, indicating high protein purity.
  • Verticillium dahliae 1 Using the biological activity detection method in Example 2, different concentrations of PevDl (22 ⁇ , ⁇ ⁇ , 5 ⁇ , ⁇ , 500 ⁇ , 100 ⁇ , ⁇ ) were set, and ⁇ could cause a typical allergic reaction, and the lowest protein concentration causing an allergic reaction was 100 ⁇ .
  • ROS Reactive Oxygen Species
  • the exponentially growing tobacco suspension cells were collected by centrifugation and resuspended in buffer (175 mM mannitol, 0.5 mM calcium chloride, 0.5 mM potassium sulfate (Beijing Chemical, analytical grade), 10 mM hydroxyethylpiperazine ethanesulfonic acid (HEPES, In Sigma), pH 6.5), resuspended cells were prepared at 0.1 g/mL (cell wet weight/volume) and incubated for 1 h at 24 ° C and 150 rpm.
  • buffer 175 mM mannitol, 0.5 mM calcium chloride, 0.5 mM potassium sulfate (Beijing Chemical, analytical grade), 10 mM hydroxyethylpiperazine ethanesulfonic acid (HEPES, In Sigma), pH 6.5
  • resuspended cells were prepared at 0.1 g/mL (cell wet weight/volume) and incubated for 1 h
  • HEPES buffer (175 mM mannitol, 0.5 mM calcium chloride, 0.5 mM potassium sulfate (Beijing Chemical, analytical grade), 10 mM hydroxyethylpiperazine ethanesulfonic acid (HEPES, Sigma), 0.3 mM.
  • Luminescent ammonia 175 mM mannitol, 0.5 mM calcium chloride, 0.5 mM potassium sulfate (Beijing Chemical, analytical grade), 10 mM hydroxyethylpiperazine ethanesulfonic acid (HEPES, Sigma), 0.3 mM.
  • PevDl was added at the same time to a final concentration of 1 ⁇ M.
  • the chemiluminescence detector Promega was used to detect the reactive oxygen species produced by the cells.
  • the leaf treatment method is the leaf treatment method in the biological activity detection in Example 2, the PevDl concentration is 1 ⁇ , and the treatment is for 24 hours.
  • the treated leaves were washed with distilled water and placed in a 2 mL tube.
  • the appropriate amount of DAB (3,3 '-Diaminbenzidine) (Sigma) solution (1 mg/mL, pH 3.8) was used to adjust the pH with NaOH. Values were added to 5.8, and pretreated plant tissues were added separately and stored at room temperature for 8 hours in the dark.
  • the above-mentioned O. lg/mL resuspended cells were transferred to a new tobacco suspension cell culture medium, cultured at 25 ° C, 150 r / min for 3 days, and then added with the protein elicitor PevDl to a final concentration of 1 ⁇ . Hey, continue to shake. Thereafter, the pH of the medium was measured with a pH meter every 10 minutes until 100 minutes after the treatment.
  • the leaf treatment method is the leaf treatment method in the biological activity detection in Example 2, the leaves treated with 1 ⁇ of PevDl for 24 hours, and the leaves are placed in a buffer (1% glutaraldehyde, 5 mM citric acid, 90 mM phosphoric acid). Disodium hydrogen (Beijing Chemical, analytical grade), pH 7.4) overnight. Then, it was boiled in absolute ethanol for 10 min, placed in 50% ethanol, and then transferred to a buffer (67 mM dipotassium hydrogen phosphate (Beijing Chemical, analytical grade), pH 12). In the staining solution (0.1% aniline blue (Sigma), 67 mM dipotassium hydrogen phosphate (Beijing Chemical, analytical grade), pH 12), stained for 1 h, placed in 70% glycerol fixed for observation.
  • a buffer 1% glutaraldehyde, 5 mM citric acid, 90 mM phosphoric acid.
  • Disodium hydrogen (Beijing Chemical, analytical grade), pH 7.4) overnight
  • the purified PevDl protein was detected by two-dimensional electrophoresis, and the single point of the protein was cut out by 10 ng/ ⁇ 1 sequencing grade modified trypsin (Promega) 30 ° C, pH 7.5 enzymatic hydrolysis 30 min ⁇ lh, ESI-MS / MS is a Q-TOF2 orthogonal accelerated electrospray ionization tandem mass spectrometer from Micromass, UK. It is equipped with a nanoliter spray source. All measurements were performed in positive ion mode. The mass spectrometer was calibrated with Glu-fib tandem mass spectrometry and the mass accuracy was less than 0.1 Da.
  • the atomizing gas is nitrogen and the collision gas is argon.
  • the source temperature is 80 ° C and the cone voltage is 50V.
  • the TOF acceleration voltage is 9.1kV.
  • the MCP detector voltage is 2150V.
  • multiple peptide sequences of the protein were obtained by MALDI-TOF and de novo sequencing, and the sequencing was (1) PPDP MYENIDI ADFNVRKGED GTIKYV F , ( 2 ) DADGLLC EAQ PGLPSN VIT. See Figure 1.
  • Primers were designed based on the results of mass spectrometry sequencing and codon degeneracy, PevDl-positive 5 '- ATGCAGTTCACCCTYGCCG-3 ' and PevDl-reverse 5 '-TTAAGCCTSGGCGGGAGC-3, and mRNA extracted with D.
  • HI-PCR was performed using HiFi reverse transcriptase polymerase (94 ° C 3 min; 94 ° C 30 s, 67 ° C 30 s, 72 ° C lmin, 35 cycles; 72 ° C lOmin; 4 ° C ° , amplification a 468 bp DNA fragment, This includes a complete open reading frame of 468 bp, which is named pevDl gene, and the sequence of this gene is shown in SEQ ID NO: 1.
  • the expression vector obtained in the step (1) was transformed into Escherichia coli BL21 (DE3) by thermal excitation. Plasmid extraction and transformation The expression of the host strain is the same as (1), and the expression is correct after correct verification. The correct recombinant expression strain was verified to be activated overnight, while the pET28a empty plasmid strain was taken as a control.
  • RESULTS A fusion-expressed protein (His-PevDl) with a molecular weight of approximately 20-kDa was obtained by SDS-PAGE and was consistent with the prediction.
  • Purification of the recombinant protein was carried out using an AKTA explorerlO protein purifier.
  • the Hitrap chelating Column was used for affinity chromatography.
  • the affinity column was first equilibrated with the washing buffer.
  • the step (2) was used to inject 5 mL of the recombinant protein solution after centrifugation at a flow rate of 1 mL/min. After elution to baseline, elute.
  • the buffer was eluted; each of the collected eluted peaks was dialyzed against a large volume of phosphate buffer at 4 ° C overnight, followed by SDS-PAGE electrophoresis and Coomassie blue staining to determine the purity of the protein.
  • Biological Activity Assay The activity of the recombinant protein elicitor on tobacco was determined by the method of Example 2.
  • the recombinant protein was observed to induce accumulation of H 2 0 2 on the treated leaves; as in the method of (2) of Example 3, it was observed that the recombinant protein caused alkalization of the tobacco cell culture medium.
  • Recombinant protein can cause allergic reaction in tobacco leaves; His-PevDl can accumulate H 2 0 2 to produce reddish brown precipitate; His-PevDl alkalizes the treated tobacco suspension cell culture medium. The highest pH of the base is consistent with that caused by naturally purified PevD 1 .
  • the first strand cDNA was synthesized using TransGen's TransScript Two-Step RT-PCR Kit. 500 ng of total RNA was used, according to the kit instructions, using TransScript RT/RI Enzyme Mix, using Anchored 01igo (dT) 18 as primer, incubating at 42 °C for 30 min, and heating at 85 °C for 5 min to inactivate the enzyme.
  • His-PevD1 could induce the expression of resistance-related genes 2 days after treatment of tobacco leaves, except for ACS1, which was not expressed until the end.
  • TMV tobacco anti-TMV
  • 50 L of the purified His-Pe V Dl (l M) solution was passed through a needleless syringe and injected into the tobacco leaves from the back.
  • Tris-HCL buffer was used as a control, and 10 strains per treatment were repeated 3 times.
  • TMV was inoculated in the uninduced upper lobe. After 1, 2, 3, 4, and 5 days of inoculation, the number and diameter of the plaque on the control and treated leaves were investigated, and the inhibition rate was calculated according to the inhibition rate formula in the example (a). See Figures 2, 3.
  • the above embodiments are merely illustrative of the preferred embodiments of the present invention, and are not intended to limit the scope of the present invention.
  • Various modifications and improvements of the technical solutions of the present invention will be made by those skilled in the art without departing from the spirit of the invention. All should fall within the scope of protection defined by the claims of the present invention.
  • Industrial applicability The protein can significantly improve plant resistance, and the concentration is low. When the concentration of the fusion protein is ⁇ ⁇ ⁇ , the induction effect of the fusion protein on the tobacco leaves for 3 days is better, and the inhibition rate can reach 59.79%.
  • the protein provides a new way to improve the disease resistance of plants, and thus has broad application prospects in agricultural production.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Biomedical Technology (AREA)
  • Biochemistry (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Plant Pathology (AREA)
  • Cell Biology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Mycology (AREA)
  • Botany (AREA)
  • Peptides Or Proteins (AREA)

Description

大丽轮枝菌蛋白质激发子基因、 蛋白序列及其应用 技术领域
本发明涉及具有多于 20个氨基酸的肽、编码该肽段的核苷酸序列及其应用， 特别涉及一 种能提高植物抗性和诱导植物防御反应的、 来自于大丽轮枝菌 （Verticillium dahliae) 的蛋白 质及编码该蛋白质的核苷酸序列及其应用。 背景技术
棉花黄萎病是一种危害性极大的维管束病害。 每年给我国的棉花生产带来极大危害， 严 重影响我国棉花生产的品质和产量。棉花黄萎病菌在分类地位上属于半知菌亚门、丝胞纲、 丝 孢目、 淡色孢科、 轮枝菌属， 它分属于黑白轮枝菌和大丽轮枝菌两个种， 在我国危害最大的 是大丽轮枝菌种（Verticillium dahliae 目前对植物病害的防治主要采取化学防治和选育品种。 但是它们往往存在着无法摒除的弊端。上世纪随着生物技术的发展， 植物诱导抗性受到关注， 激发子作为诱导植物抗性的主要因子， 愈发受到关注。 因此从大丽轮枝菌中寻找出具有新活 性， 新功能的有效物质已成为国内外科学家关注的热点。
作为我国棉病主要病害的大丽轮枝菌， 其产生的蛋白质作为激发子是一种重要的效应分 子。 它通过识别并作用植物细胞表面或亚细胞组分的受体分子来传递病原菌的激发子信号， 启动植物的防卫系统， 诱导植物的局部组织产生过敏性细胞坏死， 并通过局部细胞信号物质 的系统传递使植物最终产生系统获得性抗性。
世界上研究的大丽轮枝菌中的激发子， 主要起到毒素作用。 目前已经从大丽轮枝菌中分 离出许多能够引起植物抗病反应或致使植物发病的活性成分。如早在 1982年 Buchner, V.等就 在大丽轮枝菌的培养液中分离到一种脂多糖复合物 PLPC ( protein-lipopolysaccharide complex ) , 可以诱发与黄萎病菌类似的致病症状 （Buchner, V.等， Physiol. Mol. Plant Path.,1989,35:253-269); 同时 Kazakov, I .等用黄萎毒素处理棉苗， 显微观察显示其维管系 统产生了胶滞体与侵填体（Kazakov, I .等， Uzbekistan Biologij a Zurnali. 1989, (5):8-10)o Davis, D. A.等从大丽轮枝菌发酵液中分离到一个约 65-kDa的糖蛋白，该糖蛋白的蛋白成分能有效地 积累植保素，是主要活性成分，糖基起着结构作用（Davis, D. A.等， Physiological and Molecular Plant Pathology， 1998， 52, 259-273 )„
中国上海植物生理研究所储昭庆等人分离了一个约 26-kDa 的分泌型糖蛋白复合物,该糖 蛋白能够诱导海岛棉培养细胞中棉酚等倍半萜的合成,棉酚的积累随着糖蛋白浓度成正比，但 当该糖蛋白浓度达到较高的水平时会引起植物细胞死亡 (储昭庆等， 植物学报 1999 , 41 (9) :972〜976)。 上海植物生理研究所陈晓亚等从所制备的大丽轮枝菌 EST序列中， 获得了一 个 233个氨基酸残基组成的激发子蛋白。 该蛋白能够诱导活性氧的爆发和相关 PR基因的表 达， 在较低的浓度下诱导棉花悬浮细胞棉酚等倍半菇类植保素的合成 （APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2004, 70， 4989 - 4995；专利号 ZL 200310108076.8)。
所有已公开发表的文献表明， 大丽轮枝菌确实可以分泌产生激发子， 引起植物的防御反 应提高植物的抗性。 该菌产生的激发子性质和种类各不相同， 它们的蛋白质序列和基因序列 报道的很少， 所以寻找一种新的蛋白质激发子， 明确它的序列信息为揭示功能和进一步提高 植物的抗性是非常必要的。 发明内容
本发明的目的是提供一种能够提高植物抗性及诱导植物防御反应的来自于大丽轮枝菌的 分离蛋白质及编码该蛋白的多核苷酸序列及其应用。
本发明采用如下技术方案：
一种分离的蛋白质， 其氨基酸序列如 SEQ ID NO:2所示。
一种上述分离的蛋白质在提高植物抗性和诱导植物防御反应中的应用。
本发明分离的蛋白质在提高植物抗性和诱导植物防御反应中的应用， 其中所述植物为烟 草。
一种分离性的多核苷酸， 其核苷酸序列为下列所述之一：
( 1 ) 编码氨基酸序列如 SEQ ID NO:2所示的蛋白质的多核苷酸序列；
(2) 与多核苷酸序列 （1 ) 根据碱基互补配对原则互补的多核苷酸序列。
本发明分离的多核苷酸， 其中所述编码氨基酸序列如 SEQ ID NO:2所示的蛋白质的多核 苷酸序列如 SEQ ID NO: l所示。
一种载体， 其含有上述分离的多核苷酸。
一种遗传工程的宿主细胞， 其含有上述载体。
一种本发明的蛋白质制备方法， 其中， 包含下列方法：
( 1 ) 在表达条件下， 培养上述遗传工程的宿主细胞；
(2) 从培养物中分离出氨基酸序列如 SEQ ID NO:2所示的蛋白质。
本发明的蛋白质， 是指来自棉花黄萎病大丽轮枝菌 （Verticillium dahliae) 的培养液、 培 养液的上清液或菌体的蛋白质。 附图说明
图 1 为来自棉花黄萎病大丽轮枝菌 （Verticillium dahliae) 经过纯化的天然蛋白质通过 MALDI-TOF和 de novo测序获得的氨基酸序列与融合表达蛋白质氨基酸序列对比图，下划线 部分为天然蛋白经过测序获得的氨基酸序列， 整个序列为融合表达蛋白质的氨基酸序列； 图 2 为本发明中用水处理叶片 3天后摩擦接种 TMV， 接种 3天后拍摄的烟草叶片； 图 3 为本发明中用 1.0 μ Μ、 50 w L His- PevDl处理叶片 3天后摩擦接种 TMV， 接种 3 天后拍摄的烟草叶片。 具体实施方式
下面通过具体实例进一步说明本发明特点。
实施例 1 大丽轮枝菌的培养及发酵液制备
将大丽轮枝菌 Verticillium dahliae 菌株划线接种于 PDA平板， 25 °C培养 2周， 挑取菌落 边缘处接种于盛有 200mL液体培养基的 500ml三角瓶中， 其中液体培养基是指 Czapek-Dox加 1% 水解络氨酸 （Sigma), 25 °C、 150r/min培养 14天， 得到大丽轮枝菌的发酵液。
其中 PDA平板的制备方法为： 取去皮马铃薯 200克， 切成块， 加水 1000毫升煮沸 30分钟， 滤去马铃薯块， 将滤液补足至 1000毫升， 加葡萄糖 20克， 琼脂 15克， 待葡萄糖和琼脂溶化后 分装， 121 °C灭菌 30分钟。
其中 Czapek-Dox的制备方法为： 硝酸钠 3.0g， 无水磷酸氢二钾 1.0g， 七水硫酸镁 0.5g， 氯化钾 KCl 0.5g， 七水硫酸亚铁 0.01g， 蔗糖 30.0g (北京化工厂， 分析纯）， pH 5.6， 加去离 子水至 1L。
实施例 2 粗蛋白质激发子的制备和检测
取实施例 1中所得的发酵液， 在 4°C、 5000rpm条件下离心 lh或在 4°C、 13000rpm的条件下 离心 30min， 收集上清液， 用孔径 0.45mm的滤膜 （Whatman) 抽滤两次， 直至没有菌体。 加 入硫酸铵粉末,使上清液中硫酸铵终浓度为 80%来沉淀目标蛋白质， 4°C透析 48h， 最后将获得 的胞外总蛋白质溶解到 Tris-HCl缓冲液 （20mM, pH 8.0) 中， 蛋白质终浓度为 100 μ g/mL。 蛋 白质含量用 BC A™ protein assay kit ( Thermo Scientific)经 GF-M2000型酶标仪（山东高密彩虹 分析仪器有限公司） 在波长 590nm处测定。
生物活性的检测： 以生长 2个月， 具有 5-7片真叶的烟草为材料， 将粗蛋白溶液浓度稀释 到 50 y g/mL， 利用 lmL不带针头的注射器， 将 50 μ L溶液从叶子背面注射进叶片中去， 以 Tris-HCl缓冲液和 50 μ g/mL牛血清白蛋白溶液为对照。 经过 24h观察是否有坏死斑形成。
结果： 注射粗蛋白质激发子的叶片， 注射部位 4h后出现水渍斑， 6h后注射处叶片变得透 明较薄一些， 12h后可以呈现典型的过敏反应， 有坏死斑形成。 对照并无任何反应， 和正常叶 片一样。
实施例 3 蛋白质激发子的分离纯化及生物活性测定 使用 AKTA explore 10 ( GE Healthcare)蛋白质纯化仪对所得的总蛋白质进行进一步纯化， 样品首先通过 HP Q HiTrap™ 阴离子交换柱 （ GE Healthcare ) , 用 NaCl进行线性洗脱 ( 0%-100%， 30min), 获得到 6个蛋白质洗脱峰， 按照实施例 2中生物活性的检测方法对所得 各个蛋白质洗脱峰进行蛋白质激发子活性的检测， 将各洗脱峰中蛋白质浓度调整为 50 μ g/mL,结果表明，第 3个蛋白质峰，可以强烈的引起烟草的过敏反应；该蛋白质峰再通过 pheny HP疏水层析柱 （GE Healthcare) 和电洗脱的进一步纯化， 得到有活性的单一蛋白质。 该单一 蛋白质峰经 15 %变性聚丙烯酰胺凝胶电泳检测为单一带， 说明蛋白质纯度高。 该蛋白质表观 分子量为 17 kD (pl = 4.34), 将此蛋白质激发子命名为 PevDl (Protein Elicitor of
Verticillium dahliae 1 )。用实例 2中生物活性的检测方法，设置不同浓度的 PevDl (22μΜ， Ι ΙμΜ, 5μΜ， ΙμΜ, 500ηΜ, 100 ηΜ, ΙΟηΜ), ΙμΜ能够引起典型过敏性反应， 最低能引起过敏反应 的蛋白浓度为 100 ηΜ。
结果： 分离纯化的 PevDl可以使烟草形成过敏反应的坏死斑。
( 1 ) 氧爆发和 ¾02沉积
氧爆发 （Reactive Oxygen Species, ROS ) 在激发子诱导的植物抗病防卫反应中起着非常 重要的作用， 其参与调控气孔运动， 促进细胞编程性死亡， 直接或间接杀死入侵的病原菌， 作 为第二信使可在转录水平上激活和调控植物体内各种防卫相关基因的表达以及参与系统获得 性抗性（Systemic acquired resistance, SAR)的建立等。
离心收集指数增长期的烟草悬浮细胞并重新悬浮于缓冲液 （175mM 甘露醇， 0.5 mM氯 化钙， 0.5mM硫酸钾（北京化工， 分析纯）， 10mM羟乙基哌嗪乙磺酸（HEPES, Sigma), pH 6.5 ) 中， 配制成 0.1g/mL (细胞湿重 /体积） 的重悬细胞， 在 24° C ， 150 rpm的条件下孵育 lh。 然后取 250 重悬细胞加入 HEPES缓冲液 （175mM甘露醇， 0.5 mM氯化钙， 0.5mM硫 酸钾 （北京化工， 分析纯）， 10mM羟乙基哌嗪乙磺酸 （HEPES, Sigma), 0.3mM发光氨
(LuminoD ) 中， 同时加入 PevDl使其终浓度为 1 μ M， 开始用化学发光检测仪 （Promega) , 检测细胞所生成的活性氧。
叶片处理方法如实例 2中生物活性的检测中的叶片处理方法， PevDl浓度为 1 μ Μ， 处理 24h。 取处理的叶片用蒸熘水洗净后将其置于 2 mL管中,取适量 DAB ( 3,3 ' -Diaminbenzidine) ( Sigma)染液 (1 mg/mL, pH 3.8),用 NaOH调 pH值至 5.8, 分别加入预处理的植物组织,室温避 光保存 8小时。随后去除各管中染液， 加入 95% 乙醇， 沸水浴数分钟， 去除各管液体， 再加入 无水乙醇并沸水浴直至叶片绿色完全脱去为止， 再次吸出各管液体， 将去浸泡在 70%甘油中， 赶出细胞间气泡显微镜观察。
结果： PevDl处理约 30min后， 烟草悬浮细胞出现氧爆发现象； 在 PevDl处理的叶片部位 有明显的褐色沉积物质出现， 并且处理部位的气孔上的保卫细胞也出现红褐色沉淀。 (2) 细胞培养基的碱化
取上述 O. lg/mL的重悬细胞， 重新转接到新的烟草悬浮细胞培养基中， 25 °C， 150r/min培 养 3天后， 加入蛋白质激发子 PevDl处理， 使其终浓度为 1 μ Μ， 继续摇培。 此后每过 lOmin用 pH计测定培养基的 pH， 直到处理后 100min。
结果： 蛋白质激发子 PevDl能够引起细胞培养基的碱化， 在处理后 50min培养基的 pH达到 最高。
( 3 ) 胼胝质的沉积、 酚类物质和木质素的积累
叶片处理方法如实例 2中生物活性的检测中的叶片处理方法， 经 1 μ Μ 的 PevDl处理 24h 后的叶片， 将叶片放入缓冲液 （1%戊二醛， 5 mM柠檬酸， 90 mM磷酸氢二钠 （北京化工， 分析纯）， pH 7.4)过夜。 然后放入无水乙醇中煮 10min， 在放入 50%乙醇中， 接着转入缓冲液 ( 67mM磷酸氢二钾（北京化工， 分析纯）， pH 12)。在染色液（0.1%苯胺蓝（Sigma), 67mM 磷酸氢二钾 （北京化工， 分析纯）， pH 12) 染色 lh， 放入 70%甘油固定观察。
取 0.5g烟草细胞， 加入 PevDl使其终浓度为 1 μ M， 处理 108h后， 用 6mol/L的 2.5% w/v间 苯三酚 （Sigma) 处理， 立即观察。
取 300 L烟草悬浮细胞， 加入 PevDl使其终浓度为 1 μ Μ， 处理 108h后， 在紫外荧光显微 镜下观察酚类物质的积累。
结果： 在 PevDl处理的叶子， 细胞壁出现胼胝质星状的积累； 处理过的烟草悬浮细胞有 木质素的积累； 可以观察到酚类物质在细胞上的沉积。
实施例 4 蛋白质激发子 PevDl肽段序列的获得
纯化的 PevDl蛋白质经双向电泳检测， 切取蛋白质单点分别经 lOng/ μ 1 测序级修饰胰蛋 白质酶（ Sequencing Grade Modified Trypsin, Promega) 30°C、 pH7.5酶解 30min~lh， ESI-MS/MS 为英国 Micromass公司的 Q-TOF2正交加速电喷雾串联质谱仪。 配备纳升喷雾源。 所有测定均 在正离子方式下进行。 质谱仪用 Glu-fib的串联质谱碎片校正， 质量准确度小于 0.1Da。 雾化气 为氮气， 碰撞气体为氩气。 源温 80°C， 锥孔电压 50V。 TOF加速电压 9.1kV。 MCP检测器电压 为 2150V。毛细管电压 800V。最终通过 MALDI-TOF和 de novo测序获得该蛋白质的多个肽段序 列， 测序分别为 （1 ) PPDP MYENIDI ADFNVRKGED GTIKYV F , ( 2 ) DADGLLC EAQ PGLPSN VIT。 见附图 1。
实施例 5 蛋白质激发子 PevDl编码基因的克隆
根据质谱测序结果和密码子的简并性设计引物， PevDl-正向 5 ' - ATGCAGTTCACCCTYGCCG-3 ' 禾口 PevDl-反向 5 ' -TTAAGCCTSGGCGGGAGC-3，， 并以大 丽轮枝菌株抽提的 mRNA为模板， 选用 HiFi 反转录聚合酶进行 PT-PCR ( 94° C 3min; 94° C 30s, 67° C 30s， 72° C lmin， 35循环； 72° C lOmin; 4° C ° ，扩增出 468bp的 DNA片段， 其中包括 468bp的完整开放阅读框， 将其命名为 pevDl基因， 该基因的顺序如 SEQ ID NO: 1所 示。
实施例 6 pevDl基因的原核表达和纯化
( 1 ) 表达载体的构建
设计蛋白质激发子基因 pevDl的特异性引物， 引入带有酶切保护碱基的酶切位点， 正向 引物： 5 ' - CCGGAATTCATGCAGTTCACCCTCGCCG-3， , (下划线表示 £co RI的酶切序列）， 反向引物： 5 ' -CCCAAGCTTTTAAGCCTCGGCGGGAGC-3， , (下划线表示 H «iflII的酶切序 列）， 扩增全长 pevDl基因 ( 94° C 3min; 94° C 30s, 67° C 30s, 72° C lmin, 35个循环; 72° C lOmin; 4° C °^)0 先将 pevDl基因目的片段克隆到 pMD18-T simple载体， 经 Eco RI和 Hindlll酶切后， 将 pevDl片段克隆到 pET-28a(+)载体 (Novagen)的 Eco RI/HindIII位点， 热激发 转化到大肠杆菌 Trans lOCTransgene) ,挑取阳性克隆， 摇菌并提取质粒， 酶切并测序验证。 （2) 诱导表达
将步骤（1 )中所获得表达载体用热激发转化到大肠杆菌 BL21 (DE3)中。质粒提取及转化 表达宿主菌株的方法同 （1 )， 验证正确后， 进行表达。 将验证正确的重组表达菌株过夜活化， 同时取 pET28a空质粒的菌株作为对照。 分别取 lmL过夜培养液加入到含有 100 μ g/mL Kan的 lOOmL LB液体培养基中 （1%接种量）， 37°C , 200r/min振荡培养 2〜3h， 培养至培养液 OD600 为 0.6〜0.8时加入诱导剂 IPTG (终浓度为 0.1mmol/L)， 22.5 V , 220r/min继续振荡培养 24h， 诱 导表达目的蛋白。 高速离心， 收集菌体加入缓冲液。 经超声破碎菌体后， 4°C高速离心， 收集 上清。取 20 μ L上清液，加入 5 μ L 5 X SDS上样缓冲液（变性）重悬菌体，沸水浴中加热 10min， 13000 r/min离心 10min， 取上清进行 SDS-PAGE检测， 考马斯亮兰 R250染色， 观察表达情况。
结果： 经过 SDS-PAGE检测， 获得一个分子量约 20-kDa的融合表达蛋白 （His- PevDl ), 与预测一致。
( 3 ) 重组蛋白的纯化
利用 AKTA explorerlO蛋白纯化仪进行重组蛋白的纯化。 选用 Hitrap chelating Column柱进 行亲和层析， 先用清洗缓冲液平衡亲和柱； 取步骤 （2) 离心后的重组蛋白液 5mL进样， 流速 为 lmL/min, 淋洗至基线后， 洗脱缓冲液进行洗脱； 收集的各洗脱峰， 在大量体积的磷酸盐 缓冲液中 4°C透析过夜， 随后进行 SDS-PAGE电泳和考马斯亮兰染色检测蛋白的纯度。
结果： 获得纯化的约 20KD的融合表达蛋白 （His- PevDl重组蛋白）
实施例 7 重组蛋白 (His- PevDl)在烟草上引起的反应
生物活性测定： 采用实施例 2中方法测定重组蛋白质激发子对烟草的活性。
用 lmL不带针头的注射器， 分别将约 50 mL、 Ι μ Μ 融合表达蛋白 (His- PevDl), 相同浓 度的 pET-28a(+)空质粒表达蛋白及 Tris-HCl (20mM ,ρΗ 8.0 )溶液分别从叶子背面分别注入不 同的烟草叶片中。 24h后观察叶片上的反应。
如实施例 3中 （1 ) 的方法， 观察重组蛋白诱导 H202在处理叶片上的积累； 如实例 3中 （2) 的方法， 观察重组蛋白引起烟草细胞培养基的碱化。
结果： 重组蛋白 (His- PevDl)能够引起烟草叶片发生过敏反应； His-PevDl使处理叶片获 得 H202积累， 产生红褐色沉淀； His-PevDl使处理的烟草悬浮细胞培养基碱化， 培养基 pH最 高的时间和天然纯化的 PevD 1引起的基本一致。
( 1 ) 抗性相关基因半定量 RT-PCR分析
取所需的样品液氮研磨成粉， 按 50-100mg/mL加入 Trizol, 室温 5min静置， 12000rpm离心 5min, 弃沉淀； 按 200 μ L氯仿 / mL Trizol,震荡混匀 15min放置， 4V , 12000rpm离心 15min， 吸取上层， 加入 0.5 mL异丙醇 / mL Trizol, 混匀， -20°C静置 20min; 离心， 75%乙醇漂洗， 离 心去除乙醇， 5-10min晾干。 加入 DEPC处理水， 测 RNA浓度， 调成相同浓度。
第一链 cDNA的合成， 采用 TransGen公司的 TransScript Two-Step RT-PCR Kit。 取 500ng总 RNA,按照试剂盒说明书，用 TransScript RT/RI Enzyme Mix ,以 Anchored 01igo( dT) 18为引物 ,42 °C孵育 30min， 85 °C加热 5min使酶失活。取 1 μ L反转录产物做 PCR, β -actin为内标基因， 以抗 性相关基因特异引物做 PCR(PRl-a 基因 F:5 ' -TGGATGCCCATAACACAGC -3 ' ， R:5 ' - AATCGCCACTTCCCTCAG -3 ' ； PRl-b 基因 F:5 ' -GATGTGGGTTGATGAGAAGC -3 ' ， R:5 ' -CTCCAATTACCAGGTGGATC-3 ' ； PDF 1.2 基 因 F:5 ' -GGAAATGGCAAACTCCATGCG-3 ' ， R:5 ' - ATCCTTCGGTCAGACAAACG-3 ' ； PR1基 因 F:5 ' -ACATCAGCGGAAGCAGTAG -3 ' ， R:5 ' -GTCGGCGAAGTAGTCAAAC -3 ' ； ACS1 基 因 F:5 ' -GAGAATGAGAAGAACAGCTCA-3 R:5 -TTCTAGCACAATTAACGACGG -3 ' )， 观察抗性相关基因的表达情况。
结果： 经过半定量 RT-PCR结果可见 His- PevDl在处理烟草叶片后 2天， 就可以诱导抗性相 关基因的表达， 但是 ACS1除外， 至始至终没有表达。
(2) 诱导减少 TMV对烟草的危害
通过预实验可知 Ι μ Μ 融合表达蛋白对烟草抗 TMV有较佳效果。将 50 L、纯化后的 His- PeVDl(l M)溶液通过无针头的注射器，从背面注入烟草叶片中。以 Tris-HCL缓冲液作对照， 每处理 10株，重复 3次。 TMV的接种方法为：取新鲜 TMV病叶加 0.01 mol/L磷酸缓冲液（pH=7.4) 研磨成匀浆， 病汁液浓度为每克病叶加 15mL磷酸缓冲液。 分别确定最佳接种时间和最佳诱 导时间
( a) 最佳诱导时间
分别于融合表达蛋白处理烟草叶片后第 1、 3、 5和 7天， 在处理叶的上一位叶摩擦接种 TMV。 于接种后 3天， 调查枯斑数量， 计算枯斑抑制率。 抑制率(％)=[ (坏死斑个数 /直径 -处理组坏死斑个数 /直径) /坏死斑个数 /直径] X 100 结果如下表。
Figure imgf000009_0001
* 表中不同字母表示在 P <0.01水平时的差异显著性。
(b) 最佳接种时间
融合表达蛋白 (His- PevDl)诱导烟草 3d后， 在未诱导的上位叶接种 TMV。 接种 1,2,3,4,5天 后调查对照及处理叶片上的枯斑枯斑数目和直径，均按本实例（a)中抑制率公式计算抑制率。 见附图 2、 3。
有关具体的坏死斑个数和坏死斑直径抑制率， 结果如下表。
Figure imgf000009_0002
* 表中不同字母表示在 P <0.05水平时的差异显著性。
上述结果表明， 重组蛋白质激发子 His- PevDl可以诱导减少 TMV对烟草的危害， 在蛋白 诱导 3d后， 其诱导效果更佳， 可以达到 59.79%的抑制率； 接种 TMV 3天后的诱导效果最佳， 使坏死斑数抑制率为 68.57%， 坏死斑直径抑制率为 27.16%。
以上实施例仅是对本发明的优选实施方式进行描述， 并非对本发明的范围进行限定， 在 不脱离本发明设计精神的前提下， 本领域普通技术人员对本发明的技术方案作出的各种变形 和改进， 均应落入本发明权利要求书确定的保护范围内。 工业实用性 该蛋白质可以明显提高植物抗性， 浓度低起效快， 融合表达蛋白接种浓度为 ΐ μ Μ时， 在 融合表达蛋白处理烟草叶片 3d后， 其诱导效果更佳， 可以达到 59.79%的抑制率； 融合表达蛋 白处理烟草叶片 3d后再接种 TMV 3天后的诱导效果最佳， 使坏死斑数抑制率为 68.57%， 坏死 斑直径抑制率为 27.16%。 该蛋白质为提高植物的抗病性提供了新的途径， 因而在农业生产上 具有广阔的应用前景。

Claims

权 利 要 求
1.一种分离的蛋白质， 其特征在于， 其氨基酸序列如 SEQ ID NO:2所示。
2. 根据权利要求 1所述的蛋白质在提高植物抗性和诱导植物防御反应中的应用。
3. 根据权利要求 2所述的蛋白质在提高植物抗性和诱导植物防御反应中的应用， 其特征 在于， 所述的植物为烟草。
4.一种多核苷酸， 其特征在于， 它包含一核苷酸序列， 该核苷酸序列选自下组：
( 1 ) 编码如权利要求 1所述蛋白质的多核苷酸；
(2) 与多核苷酸 （1 ) 互补的多核苷酸。
5.根据权利要求 4所述的多核苷酸，其特征在于，其多核苷酸序列如 SEQ ID NC 所示。
6.—种载体， 其特征在于， 它含有权利要求 4或 5所述的多核苷酸序列。
7.—种遗传工程的宿主细胞， 其特征在于， 它含有权利要求 6所述的载体。
8.—种权利要求 1所述的蛋白质制备方法， 其特征在于， 包含下列方法：
( 1 ) 在表达条件下， 培养权利要求 7所述的宿主细胞；
(2) 从培养物中分离出权利要求 1所述的蛋白质。
PCT/CN2011/077486 2010-12-17 2011-07-22 大丽轮枝菌蛋白质激发子基因、蛋白序列及其应用 WO2012079370A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201010593488.5 2010-12-17
CN201010593488.5A CN102558320B (zh) 2010-12-17 2010-12-17 大丽轮枝菌分离的蛋白质多核苷酸及其应用

Publications (1)

Publication Number Publication Date
WO2012079370A1 true WO2012079370A1 (zh) 2012-06-21

Family

ID=46244044

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2011/077486 WO2012079370A1 (zh) 2010-12-17 2011-07-22 大丽轮枝菌蛋白质激发子基因、蛋白序列及其应用

Country Status (2)

Country Link
CN (1) CN102558320B (zh)
WO (1) WO2012079370A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111771911A (zh) * 2020-06-30 2020-10-16 武汉合缘绿色生物股份有限公司 一种防治烟草花叶病毒的微生物制剂及其制备方法
CN112125963A (zh) * 2019-06-24 2020-12-25 北京市农林科学院 可可毛色二孢菌LtALTA1基因及其用途

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10806145B2 (en) 2015-10-22 2020-10-20 Pherobio Technology Co., Ltd Applications of protein VdAL in improving output, product quality and drought resistance of plant and in improving fruit coloring of plant
CN105218649B (zh) * 2015-10-22 2019-01-01 北京中捷四方生物科技股份有限公司 蛋白质VdAL在提高植物产量和促进植物生长中的应用
CN108719337B (zh) * 2018-05-30 2021-03-30 崔明华 一种治愈植物癌症枯黄萎病的药物及其生产方法

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1609120A (zh) * 2003-10-22 2005-04-27 中国科学院上海生命科学研究院 棉花黄萎病菌分泌型激发子基因及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN100368432C (zh) * 2005-04-15 2008-02-13 华南农业大学 一种新的稻瘟病菌糖蛋白激发子及其纯化方法

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1609120A (zh) * 2003-10-22 2005-04-27 中国科学院上海生命科学研究院 棉花黄萎病菌分泌型激发子基因及其应用

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
WANG, BINNAN ET AL.: "The purification and characterization of a novel hypersensitive-like response-inducing elicitor from Verticillium dahliae that induces resistance responses in tobacco.", APPL MICROBIOL BIOTECHNOL., 21 June 2011 (2011-06-21) *
ZHANG, ZHIGANG ET AL.: "Effect of Protein Elicitor from Alterneria tenuissima on Cotton Resistance to Verticillium dahliae and Activitie S of Relative Enzyme.", CHINESE JOURNAL OF BIOLOGICAL CONTROL., vol. 23, no. 3, August 2007 (2007-08-01), pages 292 - 294 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112125963A (zh) * 2019-06-24 2020-12-25 北京市农林科学院 可可毛色二孢菌LtALTA1基因及其用途
CN112125963B (zh) * 2019-06-24 2022-03-15 北京市农林科学院 可可毛色二孢菌LtALTA1基因及其用途
CN111771911A (zh) * 2020-06-30 2020-10-16 武汉合缘绿色生物股份有限公司 一种防治烟草花叶病毒的微生物制剂及其制备方法
CN111771911B (zh) * 2020-06-30 2021-06-04 武汉合缘绿色生物股份有限公司 一种防治烟草花叶病毒的微生物制剂及其制备方法

Also Published As

Publication number Publication date
CN102558320B (zh) 2014-03-26
CN102558320A (zh) 2012-07-11

Similar Documents

Publication Publication Date Title
Kim et al. Xanthomonas campestris pv. vesicatoria effector AvrBsT induces cell death in pepper, but suppresses defense responses in tomato
WO2012079370A1 (zh) 大丽轮枝菌蛋白质激发子基因、蛋白序列及其应用
KR20210052449A (ko) 재조합 단백질 변이체
KR101957550B1 (ko) 항진균성 식물 단백질, 펩티드 및 사용 방법
KR102224897B1 (ko) 신규한 폴리펩타이드 및 이를 포함하는 그람음성균에 대한 항생제
Lam et al. Acaconin, a chitinase-like antifungal protein with cytotoxic and anti-HIV-1 reverse transcriptase activities from Acacia confusa seeds
WO2024037548A1 (zh) 一种植物免疫激活蛋白PmSCR1及其应用
Wu et al. GroEL protein from the potential biocontrol agent Rhodopseudomonas palustris enhances resistance to rice blast disease
CN109305996B (zh) 禾谷镰刀菌分泌型蛋白激发子FgHrip1及其应用
KR102146468B1 (ko) 항균 활성이 증가한 항균 펩타이드 및 이를 유효성분으로 함유하는 항균용 조성물
WO2019201059A1 (zh) 调控抗铝毒转录因子stop1蛋白的基因及其应用
CN113929755B (zh) 一种葡萄霜霉菌分泌的植物免疫激活蛋白及引物和应用
CN106432453A (zh) 一种烟草凝集素蛋白及其编码基因和应用
Qian et al. Preparation of a polyclonal antibody against hypericin synthase and localization of the enzyme in red-pigmented Hypericum perforatum L. plantlets
CN107446946B (zh) 禾本科植物抗病调控的酯酶基因
Seng et al. Silencing GhCOI1 in Gladiolus hybridus increases susceptibility to Alternaria brassicicola and impairs inducible defenses
WO2013123625A1 (zh) 枣树谷胱甘肽过氧化物酶基因
CN114507674A (zh) 茶树昼夜节律基因lux在提高植物抗寒性上的应用
CN108823182A (zh) 普通菜豆镰孢菌枯萎病抗病相关蛋白PvMES1、编码基因及应用
JP2000342262A (ja) 耐病性遺伝子
CN116987161B (zh) 一种弱毒变黑轮枝菌外泌蛋白激发子及其应用
CN110776571A (zh) 一种金属硫蛋白融合蛋白构建、固定化载体快速制备及其在重金属离子去除中的应用
CN115029373B (zh) 绿僵菌cfem85蛋白的应用及提高植物对灰霉菌以及蚜虫的抗性的方法
KR100328507B1 (ko) 나팔꽃 종자로부터 유래된 항균성 펩타이드의 ｃＤＮＡ를 포함하는 재조합 식물체 발현벡터
CN109721647B (zh) 一种诱导水稻免疫反应的稻瘟菌分泌蛋白及其应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 11849183

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 11849183

Country of ref document: EP

Kind code of ref document: A1