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WO2021104542A1 - Utilisation du gène osatl15 dans le riz dans le réglage de l'absorption et du transport de pesticides - Google Patents

Utilisation du gène osatl15 dans le riz dans le réglage de l'absorption et du transport de pesticides Download PDF

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WO2021104542A1
WO2021104542A1 PCT/CN2021/073421 CN2021073421W WO2021104542A1 WO 2021104542 A1 WO2021104542 A1 WO 2021104542A1 CN 2021073421 W CN2021073421 W CN 2021073421W WO 2021104542 A1 WO2021104542 A1 WO 2021104542A1
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rice
osatl15
gene
pesticides
seq
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徐汉虹
林菲
肖钰艳
张汉林
李志伟
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South China Agricultural University
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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
    • 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
    • C12N15/8286—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 insect resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits

Definitions

  • the invention belongs to the field of plant genetic engineering, and particularly relates to the application of rice gene OsATL15 in regulating the absorption and transportation of pesticides.
  • Rice is the main food crop in my country and the world. Approximately 2.2 billion people in the world feed on rice, and 60% of the population in China feed on rice. However, the excessive use of pesticides in the rice production process has caused tremendous pressure on the ecological environment. It is estimated that in the current use of pesticides, only 2% of the pesticides will actually stay on the surface of the plant, and only 0.1% will actually reach the target (Wang et al., 2007). This means that more than 99.9% of pesticides will enter the environment, chemical fertilizers and pesticides are inefficiently used, and most of them are lost to the soil and rivers, resulting in a decline in soil quality, water pollution and soil ecological diversity (Pimentel et al., 1992) . Therefore, enhancing the targeting of pesticides and increasing the effective utilization rate of pesticides are the main goals of current pesticide research and the necessary way to reduce the amount of pesticides and increase the efficiency.
  • the systemic insecticide has the characteristics of high efficiency, low toxicity and broad spectrum, and has a good control effect on piercing and sucking pests.
  • traditional spraying methods have caused a large amount of pesticides to be released into the environment, which not only affects pollinators, but also causes economic waste. Therefore, the use of isolated and cloned rice insecticide transport genes and proteins is expected to achieve targeted accumulation and regulation of systemic pesticides, reduce environmental release, and also benefit rice breeding.
  • Pesticide transfer genes can achieve targeted accumulation of pesticides and improve the effective use of pesticides. Therefore, the use of isolated and cloned rice pesticide transfer genes and proteins is expected to achieve targeted accumulation and regulation of systemic pesticides, reduce environmental release, and at the same time be beneficial to rice breeding, and has important economic value.
  • the purpose of the present invention is to overcome the above-mentioned defects and deficiencies in the prior art, and to provide the application of rice gene OsATL15 in regulating the absorption and transportation of pesticides.
  • Another object of the present invention is to provide a molecular marker detection primer for rice gene OsATL15.
  • the application of the rice gene OsATL15 in regulating the absorption and transportation of pesticides is based on the inventor’s discovery that the OsATL15 gene can increase the absorption and transportation of pesticides by rice, increase the absorption and transportation of pesticides by rice, and reach the pests infested parts and reduce the amount of pesticides in rice fields. Usage amount; it is also possible to genetically improve the conductivity of rice varieties to pesticides, and to breed rice varieties that use pesticides efficiently.
  • the pesticide is preferably a systemic pesticide; more preferably a systemic pesticide; even more preferably a neonicotinoid insecticide, an amide insecticide, an organophosphorus insecticide, and a silkworm toxin
  • a systemic pesticide more preferably a neonicotinoid insecticide, an amide insecticide, an organophosphorus insecticide, and a silkworm toxin
  • One of insecticides or carbamate insecticides most preferably one of thiamethoxam, chlorantraniliprole, omethoate, trimethoprim or methomyl.
  • amino acid sequence of the protein encoded by the rice gene OsATL15 is the following A) or B):
  • the nucleotide sequence of the rice gene OsATL15 is any one of the following A) to E):
  • D It has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with the nucleotide sequence defined by A, and the encoding is as follows SEQ ID NO: the nucleotide sequence of the amino acid shown in 1;
  • E It has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology with the nucleotide sequence defined by B, and the encoding is as follows SEQ ID NO: The nucleotide sequence of the amino acid shown in 1.
  • a molecular marker detection primer for rice gene OsATL15 consisting of the upstream primer and the downstream primer shown in SEQ ID NO: 4 and SEQ ID NO: 5:
  • the application of the molecular marker detection primer of the rice gene OsATL15 in identifying rice varieties that efficiently use pesticides includes the following steps: using the molecular marker detection primer to amplify the germplasm genomic DNA or RNA of the rice variety to be tested , Judge the sensitivity of the target gene in response to pesticide treatment through the change of gene expression.
  • the pesticide is preferably a systemic pesticide; more preferably a systemic pesticide; even more preferably a neonicotinoid insecticide, an amide insecticide, an organophosphorus insecticide, and a silkworm toxin
  • a systemic pesticide more preferably a neonicotinoid insecticide, an amide insecticide, an organophosphorus insecticide, and a silkworm toxin
  • One of insecticides or carbamate insecticides most preferably one of thiamethoxam, chlorantraniliprole, omethoate, trimethoprim or methomyl.
  • a molecular marker detection kit for rice gene OsATL15 includes the above-mentioned molecular marker detection primer.
  • the molecular marker detection kit for the rice gene OsATL15 also includes at least one of an enzyme for PCR, water for PCR and a buffer for PCR.
  • the application of the rice gene OsATL15 in cultivating transgenic rice that efficiently utilizes pesticides includes the following steps: constructing the rice gene OsATL15 on a plant expression vector, and transferring the obtained recombinant expression vector into rice for expression to obtain the efficient use of pesticides Varieties of rice.
  • the pesticide is preferably a systemic pesticide; more preferably a systemic pesticide; even more preferably a neonicotinoid insecticide, an amide insecticide, an organophosphorus insecticide, and a silkworm toxin
  • a systemic pesticide more preferably a neonicotinoid insecticide, an amide insecticide, an organophosphorus insecticide, and a silkworm toxin
  • One of insecticides or carbamate insecticides most preferably one of thiamethoxam, chlorantraniliprole, omethoate, trimethoprim or methomyl.
  • the plant expression vector is preferably pCAMBIA2300-35S.
  • the recombinant plant expression vector obtained can be transformed into plant cells or tissues by conventional biological methods such as Agrobacterium-mediated, plant virus vector, direct DNA transformation, and electrical conduction transformation. in.
  • the present invention has the following beneficial effects:
  • the present invention uses polymerase chain reaction (PCR) to isolate and clone a gene OsATL15 from rice variety Zhonghua 11 that controls the absorption and transport of pesticides in rice.
  • PCR polymerase chain reaction
  • the application of this gene can increase the absorption and transport of pesticides by rice, and make pesticides more effective. Reach the pest-infested parts and reduce the use of pesticides in paddy fields.
  • the present invention confirms the role of the gene OsATL15 in pesticide absorption and transportation, and provides new clues for studying the mechanism of pesticide utilization by rice.
  • the molecular marker detection primer of the gene OsATL15 provided by the present invention can quickly determine the sensitivity of the target gene in response to pesticide treatment through the change of the gene expression level, and breed new varieties with high efficiency and transportability.
  • Figure 1 shows the results of real-time quantitative fluorescent PCR detection of OsATL15 gene expression in OsATL15 overexpression rice lines.
  • Figure 2 is a diagram showing the comparison of the OsATL15 gene sequence of a mutant plant in which the OsATL15 gene was knocked out by CRISPR and a wild-type plant.
  • Figure 3 shows the results of detecting the content of thiamethoxam in the root and upper part of the OsATL15 mutant rice.
  • Figure 4 shows the results of detecting the content of chlorantraniliprole in the roots and upper parts of OsATL15 mutant rice.
  • Figure 5 is a graph showing the results of detecting omethoate content in the roots and upper parts of OsATL15 mutant rice.
  • Fig. 6 is a graph showing the results of detecting the content of dimehypo in the root and upper part of the OsATL15 mutant rice.
  • Figure 7 is a graph showing the results of detecting methomyl content in the root and upper part of the OsATL15 mutant rice.
  • Figure 8 is a graph showing the results of detecting the content of thiamethoxam in rice lines overexpressing the OsATL15 gene.
  • Figure 9 is a diagram showing the results of detecting chlorantraniliprole in rice strains overexpressing the OsATL15 gene.
  • Figure 10 is a graph showing the results of detecting omethoate in rice strains overexpressing the OsATL15 gene.
  • Figure 11 is a diagram showing the results of detecting Dimehypo in rice lines overexpressing the OsATL15 gene.
  • Figure 12 is a graph showing the results of detecting methomyl in rice lines overexpressing the OsATL15 gene.
  • Figure 13 shows the results of subcellular localization of OsATL15 protein-GFP in rice protoplasts
  • Figures A, B, C and D are the results of subcellular localization of protein GFP
  • Figures E, F, G and H are fusion proteins
  • Figures A and E are the results under the bright field
  • Figures B and F are the green fluorescence under the dark field
  • Figures C and G are the red fluorescence of the membrane localization protein mCherry-1008
  • D and H are overlapping fields of view
  • scale bar 10 ⁇ m.
  • Figure 14 shows the real-time fluorescent quantitative PCR detection result of the OsATL15 gene under the condition of 100 ⁇ mol/L thiamethoxam (THX) treatment for 24 hours.
  • Figure 16 is a statistical diagram of plant height of mutant rice of OsATL15 gene.
  • Figure 17 is a statistical diagram of the root length of mutant rice of OsATL15 gene.
  • Figure 18 is a phenotype map of OsATL15 mature transgenic line
  • Figure 19 is a phenotypic statistical diagram of plant height, number of tillers, number of ears, and number of empty shells in the OsATL15 transgenic line at the mature stage.
  • the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the technical field.
  • Escherichia coli DH5 ⁇ and Agrobacterium EHA105 are commonly used strains, which are stored in most molecular biology laboratories; the rice variety is wild-type Zhonghua 11 (a publicly used rice variety, commercially available).
  • the primers used in the examples were synthesized by Shenzhen Huada Gene Company, and sequencing was performed at Shenzhen Huada Gene Company.
  • RNA OMVA R6827-01
  • gDNA-free cDNA with the nucleotides shown in SEQ ID NO: 2
  • the forward primer F1 and the reverse primer R1 were used for PCR amplification.
  • a PCR product of 1896bp was obtained.
  • the 1896bp PCR product has the nucleotides shown in SEQ ID NO: 3.
  • Use http://web.expasy.org/translate/
  • OsATL15 the amino acid sequence of the protein is shown in SEQ ID NO:1.
  • R1 5'-TCATAGGTGACGAGACTGAGGTG-3'.
  • RNA from wild-type Zhonghua 11 seedlings reverse transcription to obtain cDNA as a template (the extraction and reverse transcription methods are the same as in Example 1), and use primer 1 (ACCCGGGGATCCTCTAGAGTCGAATGGCAGATCAGAAGGTG) and primer 2 (ATGATACGAACGAAAGCTCTGCATCATAGGTGACGAGACTGAGGTG) for PCR amplification (Takara cat #R045)
  • the reaction conditions are: 1 cycle: 98°C, 3min; 32 cycles: 98°C, 30s, 58°C, 30s, 72°C, 1min; 1 cycle: 72°C, 5min; 16°C, get the belt OsATL15 gene with 15 base homologous recombination arms.
  • the above PCR product was gel-recovered, and the pCAMBIA2300-35S vector backbone (provided by Wuhan Boyuan Company), which was digested with Sal I and Pst I, was homologously recombined with the In-fusion kit (Takara cat#639648) to obtain recombination Plasmid.
  • the recombinant plasmid was sent for sequencing and named pCAMBIA1300-35S-OsATL15, which was a recombinant expression vector.
  • the recombinant expression vector pCAMBIA1300-35S-OsATL15 was electroporated into Agrobacterium EHA105 (Olivia et al., 2019) to obtain recombinant strain AGL1/pCAMBIA1300-35S-OsATL15 (plasmid was extracted from the positive clone and verified by sequencing).
  • Agrobacterium culture medium containing kanamycin 50mg/L, rifampicin 50mg/L
  • culture at 28°C 180rpm shaker for 2-3 days .
  • Agrobacterium culture medium containing kanamycin 50mg/L, rifampicin 50mg/L
  • AAM medium containing 0.1mM acetosyringone As
  • the calli were transferred to NB minimal medium containing 100 mg/L hygromycin and 400 mg/L cephalosporin for selection for 3-4 weeks (one sieve).
  • the surviving callus was transferred to a second-screen medium (NB minimal medium containing 100 mg/L hygromycin and 200 mg/L cephalosporin) for selection for 3 weeks.
  • NB minimal medium containing 100 mg/L hygromycin and 200 mg/L cephalosporin
  • Transfer the resistant callus to differentiation medium (containing 100mg/L hygromycin) for differentiation, and transfer the regenerated plants after rooting on the vigorous seedling medium containing 100mg/L hygromycin (about 3-4 weeks)
  • OsATL15 rice with T 0 generation was obtained.
  • Co-cultivation medium callus induction and subculture medium + As (0.1mM/L) + glucose (10g/L), pH 5.2.
  • Agrobacterium infection of rice callus AAM medium AA macronutrients + AA trace elements + AA amino acids + MS vitamins + hydrolyzed casein (500mg/L) + sucrose (68.5g/L) + glucose (36g/L)+ As (0.1mM), pH 5.2.
  • NB basic medium N6 macroelement + B5 trace element + B5 organic component + iron salt + hydrolyzed casein (300mg/L) + proline (500mg/L) + sucrose (30g/L) + agar (8g/L) ), pH 5.8.
  • Differentiation medium NB minimal medium + 6-BA (3 mg/L) + NAA (1 mg/L).
  • Agrobacterium medium 10g/L tryptone+10g/L yeast extract+5g/L sodium chloride+15g/L agar.
  • Extraction T 0 generation of genomic DNA transfection OsATL15 rice (OMEGA cat # D2485-02), identified PCR with primers F2 and R2 (primer sequences below), the reaction conditions were: 1 cycle: 98 °C, 3min; 32 cycles: 98°C, 30s, 60°C, 30s, 72°C, 30s; 1 cycle: 72°C, 5min; 16°C.
  • the PCR-positive ( 340bp) T 0 generation transgenic OsATL15 rice plants #1, #2, and #3 were screened.
  • R2 5'-TTCCGGAAGTGCTTGACATTGGGGA-3'.
  • Real-time quantitative fluorescent PCR was performed using Bio-Rad CFX96.
  • the PCR reaction system (20 ⁇ L) is carried out in accordance with the product instruction manual SYBR Green Real-Time PCR Master Mix reagent (Takara).
  • the specific system is as follows: 10 ⁇ L SYBR Green Real-Time PCR Master Mix, 2 ⁇ L upstream and downstream primer mixture (both upstream and downstream primer concentrations are 10 ⁇ M), 7 ⁇ L RNase-free water, 1 ⁇ L cDNA template.
  • the specific reaction procedure is as follows: enzyme heat activation at 95°C, 30s, 1 cycle; denaturation at 95°C, 5s, extension 60°C, 30s, a total of 40 cycles.
  • the primer sequence for amplifying the OsATL15 gene is:
  • OsATL15 upstream primer F 5'-TACTCGCCGCCGCCGTCATA-3' (SEQ ID NO: 4);
  • OsATL15 downstream primer R 5'-CGAGGTTGAGCGTGTTGCTGTTCC-3' (SEQ ID NO: 5).
  • the primer sequence for amplifying the internal reference UBQ2 is:
  • UBQ2 upstream primer 5’-GCATCTCTCAGCACATTCCA-3’;
  • UBQ2 downstream primer 5'-ACCACAGGTAGCAATAGGTA-3'.
  • the real-time quantitative fluorescent PCR detection results of the OsATL15 gene expression in each experimental material are shown in Figure 1.
  • the expression of the OsATL15 gene is relative. It can be seen that compared with the wild-type rice japonica rice Zhonghua 11 (WT), which is not genetically modified, The expression of OsATL15 gene in OsATL15 rice lines #1, #2 and #3 with PCR-positive T 0 generation was significantly increased at the transcription level.
  • PCR-positive T 0 generation transgenic OsATL15 rice lines #1, #2 and #3 are positive T0 transgenic OsATL15 rice lines, named OsATL15-OX1, OsATL15-OX2 and OsATL15-OX3.
  • target sequence GAGGCACGTCCTGGAGAAGGAGG.
  • the target sequence is for the OsATL15 gene and specifically inactivates the OsATL15 protein.
  • the designed target sequence was added to the pCRISPR/Cas9 system's specific sticky end adapter (F: GGCA; R: AAAC), and the complete adapter primer was synthesized.
  • F3 5’-GGCA-GAGGCACGTCCTGGAGAAGG-3’;
  • R3 5'-AAAC-CCTTCTCCAGGACGTGCCTC-3'.
  • Dilute F3 primer and R3 primer into a solution with a concentration of 10 ⁇ M take 10 ⁇ L of each and mix well, and perform annealing reaction in a PCR machine from 98°C to 22°C to make F3 primer and R3 primer complementary to form a double strand with sticky ends Small snippet.
  • the original vector pOs-sgRNA containing sg-RNA was digested with restriction endonuclease Bsa I to produce sticky ends complementary to the sticky ends of the target sequence.
  • the system for digesting the original pOs-sgRNA vector with Bsa I 10 ⁇ buffer Bsa I 2 ⁇ L, Bsa I enzyme 1 ⁇ L, pOs-sgRNA vector 4 ⁇ g, ddH 2 O to make up to 20 ⁇ L, and digestion at 37°C for 12 hours.
  • the kit (OMEGA Cat#D2500-02) was used to recover and purify the digested product through the column to obtain the digested pOs-sgRNA vector, and add sterilized ddH 2 O dissolves, and it is ready to use after measuring the concentration.
  • T4 ligase is used to connect the small double-stranded fragment in step 2) and the pOs-sgRNA vector digested in step 3) to form a complete recombinant vector containing the target sequence for the OsATL15 protein and sg-RNA.
  • the 15 ⁇ L ligation system is: 10 ⁇ T4ligation buffer 1.5 ⁇ L, double-stranded small fragment 4 ⁇ L, digested pOs-sgRNA vector 3 ⁇ L, T4 DNA ligase 1 ⁇ L, ddH 2 O make up to 15 ⁇ L, 16 °C ligation for 12 hours.
  • the ligation product was transformed into Escherichia coli DH5 ⁇ , kana-resistant LB plate was cultured overnight, and positive strains were selected for sequencing to obtain a correctly sequenced recombinant vector containing target sequence and sg-RNA.
  • LR reaction system recombinant vector containing target sequence and sg-RNA 25 -50ng, pH-Ubi-cas9-7 carrier 75ng, 5 ⁇ LR ClonaseTM buffer 1 ⁇ L, TE Buffer (pH8.0) supplemented to 4.5 ⁇ L, LRClonaseTM 0.5uL.
  • the complete recombinant vector containing OsATL15 protein target sequence-sg-RNA+Cas9 obtained in step 5) was introduced into rice callus to prepare transgenic rice, which can be used in T0 generation plants.
  • a transgenic plant with completely inactivated OsATL15 protein was obtained.
  • DNA was extracted from the transplanted transgenic plants (T 0 generation), and the target sequence was detected. A total of 32 positive plants were detected.
  • Extract DNA from the transplanted positive plants, design specific primers F4 and R4 for DNA fragments within 500 bp containing the target site to amplify the DNA fragments containing the target site, and the amplified 360 bp PCR product will be sent after purification
  • R4 5’-TGGAGCTGGTAGCCAAGAATCT-3’.
  • the mutant plants were propagated, and the plants without hygromycin, Cas9 and other transgenic elements were tested in the T1 generation transgenic segregation population.
  • the seeds were harvested from a single plant, and the loss-of-function mutants without transgenic components were obtained, and they were named Crispr. -10, Crispr-9 and Crispr-17.
  • the following pesticides were selected for the experiment: the neonicotinoid insecticide thiamethoxam, the amide insecticide chlorantraniliprole, the organophosphorus insecticide omethoate, the sandworm toxin insecticide dimethicone, amino Methomyl, a formate insecticide.
  • the Crispr-10, Crispr-9 and Crispr-17 rice strains obtained in Example 3 and the content of pesticides in the rice roots, stems and leaves of the wild-type rice Zhonghua 11 were tested.
  • the thiamethoxam original drug was dissolved in DMSO to prepare a 40 mM mother liquor and stored at 4°C for later use. Pick out the rice seedlings grown in the rice incubator for 14 days, carefully rinse the root nutrient solution, and make a group of 10 rice seedlings.
  • Example 5 The effect of OsATL15 overexpression plants on the absorption and transportation of pesticides
  • the following pesticides were selected for the experiment: the neonicotinoid insecticide thiamethoxam, the amide insecticide chlorantraniliprole, the organophosphorus insecticide omethoate, the sandworm toxin insecticide dimethicone, amino Methomyl, a formate insecticide.
  • the OX-1, OX-2 and OX-3 rice lines obtained in Example 3 and the wild-type rice Zhonghua 11 rice roots, stems and leaves were tested for pesticide content.
  • the processing method is similar to that in Example 4.
  • RNA of 15-day-old rice seedlings of Hua11 was extracted, the cDNA was obtained by reverse transcription as a template, and PCR amplification was performed to amplify the full-length ORF of OsATL15 (removing the stop codon).
  • the primers used were:
  • R5 5'-CCTTGCTCACCATCAGGATCCCTAGGTGACGAGACTGAGGTG-3'.
  • the amplified target fragments were digested and recovered, and ligated with the empty vector 322-d1-eGFPn (Beijing Huayueyang) to fuse OsATL15 with GFP.
  • the fusion vectors 322-d1-eGFPn-OsATL15 and The empty vector was transferred into rice protoplasts and cultured at room temperature for 16 hours.
  • the subcellular localization of the fusion protein OsATL15-GFP and protein GFP in rice protoplasts was observed under a laser confocal microscope. The results are shown in Figure 13, which proves that the OsATL15 protein is specifically located in the rice cell membrane.
  • thiamethoxam 100 ⁇ mol/L thiamethoxam was used to treat seedlings of wild-type Zhonghua 11 grown for 14 days, and solvent water treatment was used as a control (Control). After the treatment, the RNA was extracted, and after reverse transcription, the expression of the OsATL15 gene was detected by quantitative PCR technology. The method was the same as that in Example 2, and the experiment was set to be repeated three times.
  • the OsATL15 rice Crispr-10, Crispr-9 and Crispr-17 mutants and wild-type rice Zhonghua 11 obtained in Example 3 were planted in a rice incubator, and the phenotype was observed two weeks later. 15 strains per strain, the experiment was repeated 3 times, and the results were averaged.

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  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une utilisation d'un gène OsATL15 dans le riz. Dans la présente invention, le gène OsATL15, qui commande l'absorption et le transport de pesticides par le riz, est obtenu à partir de l'isolement et du clonage à partir de la variété de riz Zhonghua 11. La séquence nucléotidique et la protéine codée associée, et un vecteur recombinant, une cassette d'expression, une lignée cellulaire transgénique et une souche recombinante contenant le gène OsATL15 peuvent améliorer l'absorption et le transport d'un pesticide par le riz, amenant le pesticide à atteindre une partie endommagée par un organisme nuisible, et réduisant la quantité de pesticide utilisée dans une rizière. Les performances de délivrance de pesticide d'une variété de riz peuvent également être améliorées par héritage, en cultivant de manière sélective des variétés de riz ayant une utilisation efficace de pesticide. De plus, le gène OsATL15 peut participer à la régulation de la hauteur du riz, ce qui a pour effet d'améliorer les ressources du germoplasme du riz.
PCT/CN2021/073421 2019-11-25 2021-01-22 Utilisation du gène osatl15 dans le riz dans le réglage de l'absorption et du transport de pesticides Ceased WO2021104542A1 (fr)

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CN111825755B (zh) * 2020-07-27 2021-09-10 江苏省农业科学院 吸收转运新烟碱类杀虫剂的质膜内在水通道蛋白及其编码基因与应用

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