WO2022073260A1 - Application du gène slhypsys de la protéine précurseur de la systémine riche en hydroxyproline de la tomate pour améliorer la résistance des plantes au flétrissement verticillien - Google Patents

Application du gène slhypsys de la protéine précurseur de la systémine riche en hydroxyproline de la tomate pour améliorer la résistance des plantes au flétrissement verticillien Download PDF

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WO2022073260A1
WO2022073260A1 PCT/CN2020/122401 CN2020122401W WO2022073260A1 WO 2022073260 A1 WO2022073260 A1 WO 2022073260A1 CN 2020122401 W CN2020122401 W CN 2020122401W WO 2022073260 A1 WO2022073260 A1 WO 2022073260A1
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slhypsys
tomato
plants
rich
systemin
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Chinese (zh)
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李先碧
裴炎
金丹
范艳华
于晓涵
侯磊
赵娟
李美华
郑雪丽
唐梦
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西南大学
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    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture

Definitions

  • the invention relates to the field of plant biotechnology, in particular to an application of tomato-rich hydroxyproline systemin precursor protein gene SlHypSys in improving plant resistance to Verticillium wilt.
  • Plant diseases are one of the long-term natural disasters that endanger agricultural production, with an annual loss of more than 10% globally due to diseases (Feng Zhanshan et al., 2013). Plant diseases not only cause crop yield reduction, but also seriously threaten the quality and safety of agricultural products and international trade, and even cause food shortages and a series of social problems in severe cases (Punja, 2004). The harm of pathogenic bacteria not only causes direct economic losses, but also produces toxins, which brings serious food safety problems. Therefore, improving the disease resistance of crops is not only the key to solving the food problem, but also an important measure to improve food safety.
  • Verticillium wilt is a worldwide disease, and the incidence of Verticillium wilt has been reported in temperate, subtropical and tropical regions (Pegg GF, 2002). Verticillium wilt is a soil-borne vascular pathogen with facultative nutritional properties (Steven J. Kleinman et al., 2009). Pathogens mutate rapidly, with many physiological races, can survive in soil for 20 years, and can infect more than 200 plant species, all plants except monocotyledonous plants, including various vegetables, fruit trees, crops, forest trees, flowers and plants, etc. All are hosts of Verticillium wilt, and the annual crop yield loss caused by Verticillium wilt reaches billions of dollars.
  • the loss of potatoes infected with Verticillium wilt can reach more than 50%, lettuce can easily reach 100%, and cotton is in Years with severe incidence of Verticillium wilt are also likely to cause no harvest.
  • the disease loss caused by Verticillium wilt is the most serious (Pegg GF, 2002; Steven J. Kleinman et al., 2009; Agrios G, 2005 ).
  • the only cloned gene for resistance to verticillium wilt is Ve1 from tomato. This gene also exists in lettuce, but the resistance is also lost after a few years. More importantly, most crops lack the antigen against verticillium wilt. , and it is difficult to obtain resistant antigens (Steven J. Kleinman et al., 2009).
  • Plant hormones play an important role in plant defense responses, and the SA, JA and ET signaling pathways that regulate plant disease resistance have always been research hotspots.
  • the defense signal peptides for insect resistance have attracted the attention of researchers, especially the role of plant defense signal peptides in regulating plant immunity to insects and pathogens (Yube Yamaguchi et al., 2011).
  • Plant defense signal peptides are a large class of peptide hormones, one of which is derived from precursor protein peptides with N-terminal secretion signals.
  • Such peptides include the hydroxyproline-rich systemin HypSys from plants such as tobacco, tomato, and petunia.
  • Tomato hydroxyproline-rich systemin (SlHypSys) contains three hydroxyproline-rich glycopeptides, SlHypSys I, II, and III, with lengths of 20, 18, and 15 amino acids, respectively. These three mature peptides It comes from the same precursor protein, has the same function, and acts as a defense signal.
  • SlproHypSys is synthesized in the vascular parenchyma cells of leaves and localized in the cell wall matrix when tomato plants are injured, induced by systemin and methyl jasmonate (Javier, 2005).
  • Verticillium wilt is a typical vascular disease. After entering plants, it continues to infect and expand in vascular tissues. The JA signaling pathway plays an important role in the defense response of Verticillium wilt (Wei Gao et al., 2013).
  • the object of the present invention is to provide a kind of tomato rich in hydroxyproline systemin precursor protein gene SlHypSys in improving the application of plants to Verticillium wilt resistance, utilize the accumulation of SlHypSys and the infection of Verticillium wilt bacteria
  • the strategy of the present invention is proposed to use SlHypSys gene to improve the resistance of plants to Verticillium wilt.
  • the present invention provides the following technical solutions:
  • nucleotide sequence of the tomato-rich systemin precursor protein gene SlHypSys as SEQ
  • the nucleotide sequence shown in ID NO.1, or the nucleotide shown in SEQ ID NO.1 has the same function through the substitution and/or deletion and/or addition of one or several bases.
  • the plant can be other plants that can be infected with Verticillium wilt, preferably, the plant is Arabidopsis, tobacco or cotton.
  • the second object of the present invention is to provide a method for improving plant resistance to Verticillium wilt by utilizing the tomato hydroxyproline-rich systemin precursor protein gene SlHypSys.
  • the present invention provides the following technical solutions:
  • the tomato systemin-hydroxyproline-rich precursor protein gene SlHypSys was constitutively expressed in plants to obtain resistance to verticillium wilt. Verticillium wilt resistant plants;
  • the nucleotide sequence of the tomato-rich systemin precursor protein gene SlHypSys is as shown in SEQ ID NO.1, or the nucleotide shown in SEQ ID NO.1 is substituted by one or several bases and/or deleted and/or added nucleotide sequences with the same function.
  • the method for constitutively expressing the tomato systemin-rich systemin precursor protein gene SlHypSys in plants is to construct a constitutive plant expression vector containing the tomato systemin-hydroxyproline-rich precursor protein gene SlHypSys , and then obtain transgenic plants through Agrobacterium-mediated transformation.
  • the constitutive plant expression vector is the expression of the tomato hydroxyproline-rich systemin precursor protein gene SlHypSys regulated by a constitutive promoter.
  • the constitutive promoter can be any constitutive promoter that can be expressed in plants, preferably, the constitutive promoter CaMV35S promoter.
  • the constitutive plant expression vector is obtained by passing the sequence shown in SEQ ID NO.1 into the BamHI and KpnI plant expression vector pLGN (CN105671076A) to obtain the plant expression vector pLGN-35S-SlHypSys.
  • the plant can be other plants that can be infected with Verticillium wilt, such as vegetables such as eggplant, tomato, pepper, lettuce, oil plants such as rapeseed, olive oil, and ornamental plants such as red leaves; preferably, the plant is a Arabidopsis, tobacco or cotton.
  • Verticillium wilt such as vegetables such as eggplant, tomato, pepper, lettuce, oil plants such as rapeseed, olive oil, and ornamental plants such as red leaves; preferably, the plant is a Arabidopsis, tobacco or cotton.
  • SlHypSys gene (SEQ ID NO. 1) from tomato, and operably linking it with a promoter, a plant constitutively expressing the tomato hydroxyproline-rich systemin precursor protein gene SlHypSys is constructed Expression vector, transform the vector into a host to obtain a transformant, and then transfer the transformant into a plant (such as Arabidopsis, tobacco and cotton, etc.), and obtain a transgenic plant after resistance screening and a series of molecular identifications.
  • the acquisition of the SlHypSys gene, and the fusion of the promoter and the SlHypSys gene to construct an expression vector for constitutively expressing the SlHypSys gene are conventional methods in the field, and the vector used is the conventional vector used in the field of plant genetic engineering.
  • the method used for transforming the transformant into the plant is the Agrobacterium tumefaciens-mediated method, which is a commonly used method for plant transgenesis.
  • the third object of the present invention is to provide a plant expression vector with improved plant resistance to Verticillium wilt.
  • the present invention provides the following technical solutions:
  • a plant expression vector with improved plant resistance to Verticillium wilt contains an expression cassette for regulating the expression of a tomato-rich hydroxyproline systemin precursor protein gene SlHypSys by a constitutive promoter, and the tomato-rich
  • the nucleotide sequence of the hydroxyproline-containing systemin precursor protein gene SlHypSys is shown in SEQ ID NO.1, or the nucleotide shown in SEQ ID NO.1 is substituted and/or deleted by one or several bases and/or added nucleotide sequences with the same function.
  • the SlHypSys gene sequence (SEQ ID NO. 1) is operably linked with the constitutive promoter CaMV35S by using genetic engineering technology to construct a plant expression vector, and after the plants are transformed, the tomato is expressed under the action of the constitutive promoter.
  • Hydroxyproline-rich precursor protein gene SlHypSys Preferred plant expression vectors have the vector structure shown in Figure 1 .
  • the tomato hydroxyproline-rich precursor protein gene SlHypSys was positively connected to the CaMV35S promoter to form a plant expression vector pLGN-35S-SlHypSys constitutively expressing the gene.
  • the fourth object of the present invention is to provide the application of plant expression vector.
  • the present invention provides the following technical solutions:
  • the present invention first clones the hydroxyproline-rich systemin precursor protein gene SlHypSys from tomato, adopts the method of molecular biology to construct a plant expression vector constitutively expressed by SlHypSys, and then utilizes the gene Through engineering method, SlHypSys gene was introduced into Arabidopsis, tobacco and cotton, and transgenic Arabidopsis, tobacco and cotton lines with normal transcription and expression were obtained. When the disease index of wild-type Arabidopsis was 50.69, the disease index of transgenic Arabidopsis was only 13.52.
  • the disease index of wild-type tobacco was 50.85, the disease index of transgenic tobacco was only 9.11; when the disease index of wild-type cotton was 53.43, the disease index of transgenic cotton was only 17.22.
  • the disease index of the transgenic plants was significantly lower than that of the wild-type control, and the resistance to Verticillium wilt was significantly improved.
  • the invention is of great significance for promoting the application of SlHypSys gene in plant disease resistance genetic engineering.
  • Figure 1 is the map of the plant expression vector pLGN-35S-SlHypSys.
  • Figure 2 shows the transcriptional expression levels of SlHypSys genes in transgenic Arabidopsis (SlHypSys-2, SlHypSys-3, SlHypSys-6, SlHypSys-12 and SlHypSys-15: independent transformants of transgenic Arabidopsis; WT: Columbia ecotype wild-type thaliana Arabidopsis; values are the mean of three technical replicates, error bars are standard error (SD).
  • SD standard error
  • Fig. 3 shows that SlHypSys gene improves the resistance of Arabidopsis to Verticillium wilt (A: 14 days of Verticillium thaliana, the disease index of Arabidopsis strains; B: 14 days of inoculation with Verticillium thaliana, the disease of Arabidopsis plants .
  • Disease resistance of transgenic Arabidopsis was evaluated by disease index. Data are the mean of three replicate experiments, and error bars are SD.
  • SlHypSys-3, SlHypSys-6 and SlHypSys-15 independent transformants of transgenic Arabidopsis
  • WT Colombia ecotype wild type Arabidopsis thaliana.**: Compared with wild type Arabidopsis thaliana, the difference level of disease index reached extremely significant (p ⁇ 0.01)).
  • Fig. 4 is the expression level of SlHypSys gene transcription in transgenic tobacco (SlHypSys-1, SlHypSys-2, SlHypSys-3, SlHypSys-4, SlHypSys-12 and SlHypSys-15: transgenic tobacco independent transformants; WT: wild-type tobacco. Values are Mean of three technical replicates, error bars are SD of three replicates).
  • Figure 5 shows that SlHypSys gene improves the resistance of tobacco to Verticillium wilt (A: inoculated with Verticillium dahlia for 7 days, the disease index of tobacco lines; B: inoculated with Verticillium dahlia for 7 days, the disease of tobacco leaves; disease resistance of transgenic tobacco Sex was evaluated by disease index, values are the mean of three replicates, and error bars are SD.
  • SlHypSys-1 and SlHypSys-4 independent transformants of transgenic tobacco; WT: wild-type tobacco. **: compared with wild-type tobacco The difference level of disease index reached extremely significant (p ⁇ 0.01)).
  • Figure 6 is the transcriptional expression level of SlHypSys gene in transgenic cotton (SlHypSys-1, SlHypSys-2 and SlHypSys-5: transgenic cotton independent transformants; WT: transgenic recipient wild-type cotton; values are the average of three technical replicates, Error bars are standard error of triplicate).
  • SlHypSys transgenic cotton improves the resistance to Verticillium wilt (A: disease index of transgenic cotton leaves inoculated with Verticillium wilt for 5 days; B: disease of cotton leaves after inoculated with Verticillium wilt for 5 days; disease resistance of transgenic cotton Disease index was evaluated. Data are the mean of three replicate experiments, and error bars are SD. SlHypSys-1, SlHypSys-2 and SlHypSys-5: transgenic cotton independent transformants. WT: gene recipient wild-type cotton.** : Compared with wild-type cotton, the difference level of disease index reached extremely significant (p ⁇ 0.01)).
  • RNA solution Take 7 ⁇ L of the above extracted RNA solution, add 1 ⁇ L RNase-free DNase, 2 ⁇ L RNase-free DNase buffer, mix well, react at 42°C for 2 min in the PCR machine, then add 4 ⁇ L reverse transcriptase buffer, 1 ⁇ L reverse transcriptase buffer. Transcriptase, 1 ⁇ L reverse transcription primer mix, 4 ⁇ L double-distilled water without RNase and DNase, after mixing, react at 37 °C for 15 min to synthesize cDNA, and then react at 85 °C for 5 s to terminate the reaction. The synthesized cDNA was stored at -20°C.
  • Upstream primer F-SlHypSys 5'-cgcggatccgcg atgatcagcttcttcagagc-3' (SEQ ID NO.2);
  • Downstream primer R-SlHypSys 5'-cggggtaccccg ttaataggaagcttgaagaggc-3' (SEQ ID NO.3);
  • PCR amplification was performed using PrimesSTAR MAX DNA Polymerase. Amplification procedure: pre-denaturation at 98°C for 3 min; denaturation at 98°C for 10s, annealing at 57°C for 10s, extension at 72°C for 20s, and amplification for 30 cycles.
  • the transformed engineered bacteria were added to 500 ⁇ L of LB medium, cultured at 37°C at 200 rpm for 1 h, and then spread on the Mycin LB plate, cultured at 37 °C overnight, pick a single colony and inoculate it into a test tube containing about 3 mL of LB medium, shake at 37 °C and 200 rpm overnight, take an appropriate amount of bacterial liquid and send it to a sequencing company for sequencing, positive cloning projects containing the target fragment
  • the bacteria were stored at -80°C.
  • Embodiment 2 the construction of the plant expression vector that constitutively expresses SlHypSys gene and the acquisition of engineering bacteria
  • the above-mentioned cloned vector engineering bacterial plasmids verified to be correct by sequencing were extracted, and double-enzyme digested with BamHI and KpnI.
  • the digested products were subjected to agarose gel electrophoresis, and the target fragment SlHypSys was recovered.
  • the plant expression vector pLGN was digested with BamHI and KpnI. After the digestion was completed, agarose gel electrophoresis was performed to recover large fragments.
  • the recovered gene and vector fragment were ligated by T4 DNA ligase, and the ligated product was transferred into E. coli DH5 ⁇ by heat shock method.
  • the transformed bacteria that obtained the SlHypSyss digestion fragment of the target gene was the plant expression vector pLGN-35S-SlHypSys engineering bacteria, which was stored at -80 °C.
  • the plasmids of E. coli engineering bacteria were extracted, and then Agrobacterium LBA4404 and GV3101 were transformed by electroporation.
  • the transformed bacteria were screened and cultured on YEB plates with additional Km and Sm, Km and Rif (rifampicin), and single resistant colonies were obtained. Inoculate into YEB liquid medium with additional Km and Sm, Km and Rif (rifampicin), collect the cells and extract the Agrobacterium plasmid, and then double-enzyme digestion verification with BamHI and KpnI, the correct engineering bacteria are stored in - 80°C.
  • the wild-type Arabidopsis thaliana was used as the material for genetic transformation, and the seeds after the Agrobacterium dipping were harvested after the seeds were mature.
  • the seeds harvested after the genetic transformation by the flower dip transformation method were sterilized with 75% alcohol for 15 minutes, and then evenly inoculated on the screening plate with additional 100 mg/L Km (kanamycin) for germination. If the grown seedling is green, it is Transgenic plants were transplanted into the special soil for culturing Arabidopsis when the plants had more than 2 leaves (grassy carbon soil: vermiculite: perlite in a ratio of 3:1:1), and seeds were harvested after maturity. Each plant is a transformant.
  • Arabidopsis screening medium MS inorganic+MS organic+Km 100mg/L+2.5g/L Gelrite (factor), pH6.0
  • transgenic Arabidopsis RNA was extracted and cDNA was synthesized according to the method of Example 1 using the young leaves of the transgenic plants as materials.
  • the transcriptional expression level of SlHypSys gene in transgenic Arabidopsis was detected by Real-time PCR method.
  • SlHypSys gene A specific fragment of SlHypSys gene was amplified using cDNA as template.
  • the upstream and downstream primers of the SlHypSys gene are respectively SlHypSys UP: 5'-ttaccacctccttctccc-3' (SEQ ID NO.4) and SlHypSys DN: 5'-tacataatcgtgcctcccc-3' (SEQ ID NO.5).
  • the Arabidopsis AtACT2 gene was used as the internal standard.
  • the upstream and downstream primers of the AtACT2 gene were AtACT2 UP: 5'-tatcgctgaccgtatgag-3' (SEQ ID NO.6) and AtACT2 DN: 5'-ctgagggaagcaagaatg-3' (SEQ ID NO.7).
  • the 20 ⁇ L Real-time PCR reaction system includes: 1 ⁇ L cDNA template, 1 ⁇ L upstream and downstream primers of the target gene, 10 ⁇ L 2 ⁇ iQ SYBR Green Supermix, and 7 ⁇ L ddH 2 O.
  • Real-time PCR amplification conditions pre-denaturation at 95°C for 3 min; denaturation at 94°C for 10s, annealing at 57°C for 30s, extension at 72°C for 30s, a total of 40 cycles of amplification. After amplification, the relative expression of SlHypSys gene was analyzed by Gene Study software.
  • Verticillium decidua V991 strain (Verticillium dahliae) preserved in solid PD medium (potato medium) and inoculate it into liquid PD medium, 180rpm, 26 °C of shaking culture for 7d, and then press 10% (bacteria liquid)
  • the ratio of /PD medium was inoculated into liquid PD medium, 180rpm, 26 °C shaking culture for 10d, filtered with four layers of sterile gauze to remove mycelium and impurities in the bacterial liquid, deionized water was adjusted to adjust the spore concentration to 10 8 / ml as the inoculum.
  • Arabidopsis thaliana seedlings that have been cultivated for one week are uprooted, then placed neatly in a 150mm petri dish with soil, poured into the mixed inoculum solution, the inoculation dose is 10mL/plant, soaked at room temperature for 24 hours, and then transplanted into In moist soil, 16 hours of light, 8 hours of dark culture, 20°C (dark culture)-24°C (light culture), and cultured in a light incubator with a humidity of 70%.
  • the disease grades of the plants were counted according to the standard of grade 0-4 and the disease index was calculated. Wild-type plants transformed with recipient material were used as controls.
  • grade grading standard grade 0: no disease in plant leaves; grade 1: disease in 0-25% of leaves; grade 2: disease in 25%-50% of leaves; grade 3: disease in 50%-75% of leaves; grade 4 : More than 75% of the leaves showed disease.
  • the formula for calculating the disease index :
  • Disease index ( ⁇ disease grade ⁇ number of plants ⁇ )/(4 ⁇ total number of inoculated plants) ⁇ 100
  • Embodiment 5 the genetic transformation of tobacco and the acquisition of transgenic tobacco
  • Tissue culture medium for tobacco genetic transformation (1) Tissue culture medium for tobacco genetic transformation:
  • Seed germination medium MSB (MS inorganic salt + B5 organic) + 1.0% agar powder, prepared with tap water, natural pH.
  • MSB MS inorganic salt + B5 organic
  • NAA 0.5 mg/L 6-BA + 200 ⁇ mol/L AS, pH 5.6
  • solid medium with 1.0% agar powder for solidification.
  • MSB MS inorganic salt+B5 organic
  • NAA 0.5mg/L 6-BA+1.0%
  • agar powder pH5.8.
  • Sprout induction medium MSB (MS inorganic salt + B5 organic) + 2 mg/L 6-BA + 1.0% agar powder, pH 5.8.
  • Rooting medium MSB (MS inorganic salt + B5 organic) + 1.0% agar powder, pH 6.0.
  • the recombinant Agrobacterium containing the pLGN-35S-SlHypSys plant expression vector was inoculated into the liquid YEB medium, and cultured overnight at 28°C with shaking at 200 rpm to an OD600 of 1.0-1.2. After the bacterial solution was centrifuged, the bacterial cells were collected, and the bacterial cells were resuspended in an equal volume of MSB liquid medium, and the resuspended solution was the infusion solution for transformation.
  • the leaves of tobacco sterile seedlings cultivated for 20 days were cut into leaf discs of 3-5 mm, infiltrated for 1 hr in the infusion solution, and the bacterial solution was removed. After the co-cultivation was completed, the explants were substituted into the callus induction medium supplemented with 100 mg/L kanamycin and 200 mg/L cephalosporin, and cultured in a photoperiod of 25 °C, 16 hr light/8 hr dark culture, and subcultured after 20 days. The seedling induction medium is subcultured once after 20 days, and the young shoots are produced at the edge of the leaf disc. analyze.
  • GUS staining solution 500mg/L X-Gluc, 0.1mol/L K 3 Fe(CN) 6 , 0.1mol/L K 4 Fe(CN) 6 , 1% Triton X-100(V/V), 0.01mol/L Na 2 EDTA, 0.1 mol/L phosphate buffer (pH 7.0).
  • the pLGN-35S-SlHypSys plant expression vector contains the GUS gene controlled by the CaMV35S promoter. Therefore, the transgenic plants can be rapidly identified by GUS histochemical staining. According to the method of Jefferson (1987), a little leaf tissue of Km-resistant seedlings was cut, added to GUS histochemical staining solution, stained at 37°C for 5h, and then decolorized with 95% ethanol until the green color was cleared. The last blue color is the transgenic plant, otherwise it is the non-transgenic plant.
  • SlHypSys transgenic tobacco plants used young leaves as materials, respectively extracted RNA from GUS-positive and wild-type plant leaves, and synthesized one-strand cDNA of each sample RNA according to the instructions of the cDNA one-strand synthesis kit (methods and implementation of RNA extraction and cDNA synthesis).
  • Example 1 is the same). Then a specific fragment of the SlHypSys gene was amplified using the cDNA as a template.
  • the upstream and downstream primers of the SlHypSys gene are respectively SlHypSys UP: 5'-ttaccacctccttctccc-3' (SEQ ID NO.4) and SlHypSys DN: 5'-tacataatcgtgcctccc-3' (SEQ ID NO.5).
  • the tobacco 18S gene was used as the internal standard.
  • the upstream and downstream primers of the 18S gene are respectively Nt18S UP: 5'-aggaattgacggaagggca-3' (SEQ ID NO.8) and Nt18S DN: 5'-gtgcggcccagaacatctaag-3' (SEQ ID NO.9).
  • the 20 ⁇ L Real-time PCR reaction system includes: 1 ⁇ L cDNA template, 1 ⁇ L upstream and downstream primers of the target gene, 10 ⁇ L 2 ⁇ iQ SYBR Green Supermix, and 7 ⁇ L ddH2O.
  • Verticillium wilt for inoculation of transgenic tobacco for disease resistance identification is the same as that in Example 4, and the spore concentration is adjusted to 10 10 spores/mL.
  • Tobacco plants with 7-8 true leaves cut the third to fifth leaves from the top to the bottom, wrap the petioles with moist absorbent paper, and place them in the inoculation box evenly and neatly, and gently squeeze with a pipette tip Press the junction of the main vein and the secondary vein of the leaf to achieve the purpose of artificial injury.
  • grade grading standard grade 0: no disease in leaves; grade 1: disease in 0-25% of the leaf area; grade 2: disease in 25%-50% of the leaf area; grade 3: disease in 50%-75% of the leaf area; Grade 4: Disease on more than 75% of the leaf area.
  • Disease index ( ⁇ disease grade ⁇ number of plants ⁇ )/(4 ⁇ total number of inoculated plants) ⁇ 100.
  • Tobacco leaves were inoculated with Verticillium wilt for 7 days, the disease index of wild-type plants reached 50.74, and the disease index of transgenic lines SlHypSys-1 and SlHypSys-4 were 9.11 and 15.43, respectively.
  • the T test results showed that the disease index of the transgenic line was significantly decreased compared with the disease index of the wild-type control (A in Figure 5).
  • the diseased area of wild-type leaves exceeded 50%, while the diseased area of transgenic tobacco leaves was less than 10% (Fig. 5, B).
  • the results showed that SlHypSys gene could effectively improve the resistance of tobacco to Verticillium wilt.
  • MSB MS inorganic salt + B5 organic
  • Seed germination medium 1/2MSB+20g/L sucrose+6g/L agar, prepared with tap water, natural pH;
  • Co-culture medium MSB+0.5mg/L IAA (indoleacetic acid)+0.1mg/L KT (6-furfuraminopurine)+30g/L glucose+100 ⁇ mol/L acetosyringone+2.0g/L Gelrite (Sigma ), pH 5.4;
  • Embryogenic callus induction medium MSB+0.1mg/L KT+30g/L glucose+2.0g/L Gelrite, pH5.8;
  • Liquid suspension medium MSB+0.1mg/L KT+30g/L glucose, pH5.8;
  • Somatic embryo maturation medium MSB+15g/L sucrose+15g/L glucose+0.1mg/L KT+2.5g/L Gelrite, pH6.0;
  • Seedling medium SH+0.4g/L activated carbon+20g/L sucrose, pH 6.0 (Schenk & Hildebrandt, 1972).
  • Preparation of Agrobacterium dipping solution for transformation obtain a single colony of Agrobacterium that integrates the plant expression vector pLGN-35S-SlHypSys by streaking method, then pick a single colony and inoculate it with additional 50mg/L Km and 125mg/L Sm (streptomycin).
  • liquid YEB 5g/L sucrose, 1g/L bacterial yeast extract, 10g/L bacterial tryptone, 0.5g/L MgSO 4 ⁇ 7H 2 O, pH7.0
  • the bacterial solution was inoculated into 20 mL of liquid YEB without antibiotics at a ratio of 5%, and cultured at 28° C. and 200 rpm to an OD600 of about 0.5.
  • the explants were dipped with Agrobacterium dipping solution for 20 min, the bacterial solution was poured out, and the excess bacterial solution on the surface of the explants was removed with sterile filter paper.
  • Culture for 2 days inoculate the hypocotyls into the screening degerming medium, and substitute them into the callus induction medium supplemented with kanamycin (Km) and cephalosporin (cef) after 20 days to induce callus, and subculture once every 20 days. After 60 days, the cells were subcultured into the embryogenic callus induction medium, and after the embryogenic callus was obtained, liquid suspension culture was carried out to obtain a large number of embryogenic callus with consistent growth.
  • Km kanamycin
  • cef cephalosporin
  • the embryogenic callus cultured in liquid suspension was filtered through a 30-mesh stainless steel mesh, and the embryogenic callus under the sieve was inoculated into the somatic embryo maturation medium evenly and dispersedly, and a large number of somatic embryos were produced in about 15 days, which were substituted into the SH medium, Promote the further growth of somatic embryos.
  • the regenerated seedlings with 3-4 leaves were transplanted into the greenhouse for propagation.
  • the SlHypSys gene was transferred into the cotton genome. After induction of Km-resistant callus, embryogenic callus and somatic embryos, the somatic embryos became seedlings, and then SlHypSys transgenic cotton plants were obtained.
  • SlHypSys transgenic cotton plants used young leaves as materials, respectively, extracted RNA from GUS-positive and wild-type plant leaves, and synthesized one-strand cDNA of each sample RNA according to the instructions of the cDNA one-strand synthesis kit (RNA extraction and cDNA synthesis methods were the same as the embodiment). 1), and then use cDNA as a template to amplify a specific fragment of the SlHypSys gene.
  • the upstream and downstream primers of the SlHypSys gene are respectively SlHypSys UP: 5'-TTACCACCTCCTTCTCCC-3' (SEQ ID NO.4) and SlHypSys DN: 5'-TACATAATCGTGCCTCCC-3' (SEQ ID NO.5).
  • the cotton histone GhHIS3 gene was used as the internal standard.
  • the upstream and downstream primers of the GhHIS3 gene were GhHIS3 UP: 5'-GAAGCCTCATCGATACCGTC-3' (SEQ ID NO. 10) and GhHIS3 DN: 5'-CTACCACTACCATCATGGC-3' (SEQ ID NO. 11) (Zhu YQ et al., 2003).
  • the 20 ⁇ L Real-time PCR reaction system includes: 1 ⁇ L cDNA template, 1 ⁇ L upstream and downstream primers of the target gene, 10 ⁇ L 2 ⁇ iQ SYBR Green Supermix, and 7 ⁇ L ddH2O.
  • the preparation method of the transgenic cotton for disease resistance identification of pathogenic bacteria is the same as that in Example 4.
  • grade grading standard grade 0: no disease in cotton leaves; grade 1: disease in less than 25% of the leaf area; grade 2: disease in 25%-50% of the leaf area; grade 3: disease in 50%-75% of the leaf area; Grade 4: Disease on more than 75% of the leaf area.
  • Disease index ( ⁇ disease grade ⁇ number of plants ⁇ )/(4 ⁇ total number of inoculated plants) ⁇ 100.
  • Verticillium wilt resistance identification of all transgenic plants was carried out according to the above method.
  • the results showed that the disease index of wild-type cotton was 65.83 after inoculation for 5 days, and the disease index of transgenic lines SlHypSys-1, SlHypSys-2 and SlHypSys-5 were 23.61 respectively. , 18.65 and 16.56.
  • the T-test results showed that the disease index of the transgenic cotton line was significantly lower than that of the wild-type cotton (A in Figure 7). Five days after inoculation, the leaves of the wild-type cotton showed severe disease, while the leaves of the transgenic cotton line showed only a few lesions (B in Figure 7). The results showed that SlHypSys could significantly improve the resistance of cotton to Verticillium wilt.
  • the present invention introduces the tomato-rich systemin precursor protein gene SlHypSys into wild-type Arabidopsis thaliana, tobacco and cotton by transgenic means to obtain transgenic Arabidopsis thaliana, tobacco and cotton, and its transgenic plants
  • the disease index after inoculation with Verticillium wilt was significantly lower than that of the wild-type control, thus proving that the gene can improve the resistance of Arabidopsis, tobacco and cotton to Verticillium wilt.

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Abstract

L'invention concerne une application du gène SlHypSys de la protéine précurseur de la systémine riche en hydroxyproline de la tomate pour améliorer la résistance des plantes au flétrissement verticillien. En clonant le gène de la protéine précurseur phylogénétique riche en hydroxyproline de la tomate, SlHypSys, et en utilisant des procédés de biologie moléculaire pour construire un vecteur d'expression végétale pour l'expression constitutive de SlHypSys, puis en utilisant des procédés de génie génétique pour introduire le gène SlHypSys dans une plante, on obtient des souches transgéniques d'Arabidopsis thaliana, de tabac et de coton ayant une expression transcriptionnelle normale ; les indices de maladie de toutes les plantes transgéniques obtenues sont significativement inférieurs à ceux du témoin de type sauvage, la résistance au flétrissement verticillien est significativement plus élevée, indiquant clairement que le gène SlHypSys peut être utilisé pour améliorer la résistance des plantes au flétrissement verticillien.
PCT/CN2020/122401 2020-10-09 2020-10-21 Application du gène slhypsys de la protéine précurseur de la systémine riche en hydroxyproline de la tomate pour améliorer la résistance des plantes au flétrissement verticillien WO2022073260A1 (fr)

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CN115976046A (zh) * 2022-10-19 2023-04-18 吉林大学 SlCAS基因及其所编码的蛋白质在调控番茄抗灰霉病中的应用
CN116042649A (zh) * 2022-11-11 2023-05-02 河北省农林科学院棉花研究所（河北省农林科学院特种经济作物研究所） 一种编码富含半胱氨酸的非分泌型小分子肽及其编码基因与应用
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