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WO2017128302A1 - Procédé de criblage d'un gène de résistance à un herbicide de type inhibiteur hppd et application - Google Patents

Procédé de criblage d'un gène de résistance à un herbicide de type inhibiteur hppd et application Download PDF

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Publication number
WO2017128302A1
WO2017128302A1 PCT/CN2016/072810 CN2016072810W WO2017128302A1 WO 2017128302 A1 WO2017128302 A1 WO 2017128302A1 CN 2016072810 W CN2016072810 W CN 2016072810W WO 2017128302 A1 WO2017128302 A1 WO 2017128302A1
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Prior art keywords
hppd inhibitor
hppd
screening
gene
inhibitor herbicide
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Ceased
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PCT/CN2016/072810
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English (en)
Chinese (zh)
Inventor
余宗兰
邓龙群
胥南飞
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Gevoto LLC
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Gevoto LLC
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    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • 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)

Definitions

  • the present invention relates to the field of genetic engineering, and in particular to a method and application for screening an anti-HPPD inhibitor herbicide gene.
  • HPPD 4-Hydroxyphenylpyruvate dioxygenase
  • HGA 2,5-dihydroxyphenylacetate
  • HPPD has become another new herbicide target enzyme following ALS, ACC and Protox.
  • the enzyme inhibitor has the characteristics of broad spectrum, high efficiency, low residue, good environmental compatibility and safe use in herbicidal treatment, it has attracted people's attention to the research of its inhibitor.
  • the discovery of the inhibitor has environmental protection. A major role, this is also the trend of future development of herbicide production.
  • herbicide-tolerant crops take full advantage of the advantages of herbicides.
  • herbicide-tolerant crops have brought huge benefits to farmers and the environment.
  • Glyphosate-tolerant corn and soybeans are currently the most widely produced, making glyphosate widely available.
  • glyphosate resistant weeds continue to appear, affecting the effectiveness of glyphosate resistant crops. Therefore, it is imperative to develop new herbicide-tolerant crops, such as anti-HPPD inhibitor herbicide crops.
  • the object of the present invention is to provide a method for screening anti-HPPD inhibitor herbicide gene, and screening the anti-HPPD inhibitor herbicide gene by using the method, and screening the isolated individual strains is not targeted, and the target method is stronger than the traditional method. It is simple and can screen more anti-HPPD inhibitor herbicide genes in a short time, and it is easy to screen anti-HPPD inhibitor herbicide genes from rare samples.
  • a method for screening an anti-HPPD inhibitor herbicide gene comprises the following steps:
  • a plurality of microorganisms containing HPPD inhibitor herbicide-resistant microorganisms are cultured in a resistance screening medium containing an HPPD inhibitor to obtain a resistant strain, and purified and cultured to obtain a monoclonal;
  • HPPD is 4-Hydroxyphenylpyruvate dioxygenase.
  • the present invention provides a rapid and effective method for screening high-throughput screening of anti-HPPD inhibitor herbicide genes by using the method for screening anti-HPPD inhibitors in the current anti-HPPD inhibitor gene screening method.
  • Herbicide-like genes do not need to be screened for isolated individual strains. They are more targeted and simpler to operate than traditional methods, and can be screened for more anti-HPPD inhibitor herbicide genes in a short period of time.
  • the specific process of the culture is:
  • the microbial sample is cultured 1 to 10 times in a resistance screening medium containing an HPPD inhibitor, and when the number of cultures is greater than 1 time, the HPPD inhibition of the latter culture is performed in the resistance screening medium used for each adjacent culture.
  • the concentration of the agent is not lower than the previous one, and the strain cultured in the latter culture is derived from the microbial species that survived in the previous culture.
  • the resistance verification specifically comprises:
  • the HPPD inhibitor concentration gradient is set to the highest concentration of the HPPD inhibitor concentration of the resistant strains selected in step 1), and the recombinant strain in step 3) is placed in the resistance screening medium set in the HPPD inhibitor content gradient. Culture to screen for anti-HPPD inhibitor herbicide genes.
  • the concentration of the HPPD inhibitor of the resistant strain is the highest concentration.
  • the HPPD inhibitor concentration gradient of the resistant screening medium can be set to 0.1 mM, 0.5 mM, 1 mM, 2 mM, 5 mM, 10 mM mesotrione.
  • the resistance screening medium used in step 3) may be slightly different from step 1), such as the resistance screening medium formulation provided in step 1) plus the recombinant strain Resistance to antibiotics.
  • the method for screening an anti-HPPD inhibitor herbicide gene as described above the HPPD inhibitor is mesotrione or sulfonate.
  • Mesotrione also known as mesotrione, has the molecular formula C 14 H 13 NO 7 S, molecular weight 339.32, CAS No: 104206-82-8. It is a major component of a class of commonly used HPPD inhibitor herbicides.
  • Tembotrione a triketone herbicide reported by Bayer, has the molecular formula C 17 H 16 ClF 3 O 6 S, molecular weight 440.82, CAS No: 335104-84-2.
  • step 1)
  • the HPPD inhibitor content ranges from 1 to 20 mM mesotrione or 0.25 to 5 mM sulfonone, and the spacing between adjacent concentration gradients is 1.5 to 5 times.
  • the interval between adjacent concentration gradients is 1.5 to 5 times, that is, the concentration of the HPPD inhibitor in the medium in the latter culture is 1.5 to 5 times the concentration of the HPPD inhibitor in the medium in the previous culture.
  • the method for screening an anti-HPPD inhibitor herbicide gene as described above wherein the resistance screening medium is a SMNT minimal medium containing 1-20 mM mesotrione or 0.25-5 mM cyclosulfone;
  • the SMNT minimal medium comprises the following components in parts by volume:
  • the 5 ⁇ M9 salt solution includes: Na 2 HPO 4 ⁇ 7H 2 O 60-68 g/L, KH 2 PO 4 14-16 g/L, NaCl 2.4-2.6 g/L, NH 4 Cl 4.8-5.2 g/ L;
  • the solvent used in the 5 ⁇ M9 salt solution, the MgSO 4 solution, the CaCl 2 solution, and the 0.128% tyrosine solution was water.
  • tyrosine can be utilized in the resistance screening medium to synthesize plastoquinone and tocopherol to survive.
  • 16s rRNA is ubiquitous in all bacterial chromosomal genes. rRNA is involved in the bioprotein synthesis process, its function is essential for any organism, and it is a long calendar in biological evolution. In the process, the gene sequence is conservative and the variation is small. It can be regarded as the time clock of biological evolution.
  • the variable region sequence varies from bacteria to bacteria, and the constant region sequence is basically conserved. Therefore, primers can be designed by using the constant region sequence to expand the 16s rDNA fragment. Increased, the difference between the variable region sequences is used to classify and identify bacteria of different genus and strains.
  • Primers that amplify 16s rRNA are as follows:
  • the upstream primer is 27F and the downstream primer is 1492R.
  • the plasmid is pADV3;
  • the sequence of the pADV3 was engineered from the prokaryotic expression vector pUC57 by replacing the MCS sequence in pUC57 with the sequence shown in SEQ ID NO: 1.
  • the modified pADV3 has only a partial sequence of MCS (Multiple Cloning Site).
  • MCS Multiple Cloning Site
  • pADV3 can be used with E. coli Trans1-T1.
  • Trans1-T1 competent cells are the fastest growing competent cells. On the ampicillin plate, clones can be seen from 8 to 9 hours. For blue and white spots, blue spots can be seen at 12 hours. The overnight cultured monoclonals are in 2 ml. Small amount of plasmid can be extracted in LB medium for 4 ⁇ 5h; it is suitable for efficient DNA cloning and plasmid amplification, reducing the occurrence of homologous recombination of cloned DNA, increasing the yield and quality of plasmid DNA; with T1, T5 phage resistance Sex. The transformation efficiency was up to 10 9 cfu/ ⁇ g using the pUC19 plasmid.
  • the anti-HPPD inhibitor herbicide gene obtained by the method for screening an anti-HPPD inhibitor herbicide gene as described above is used for cultivating an anti-HPPD inhibitor herbicide plant variety.
  • the method provided by the invention can screen, isolate and analyze the anti-HPPD inhibitor gene in a short time without screening the isolated individual strains, and has higher target and simple operation than the traditional method, and saves a lot of manpower and material resources.
  • the method provided by the present invention can screen and isolate microorganisms and genes thereof against HPPD inhibitors from rare samples having extremely low levels of resistant microorganisms.
  • strains with different concentration resistance can be isolated and have a broad application prospect.
  • Figure 1 is a graph showing the color change of Trans1-T1 E. coli transformed with pADV3-pfHPPD in the medium in Example 5.
  • a method for screening an anti-HPPD inhibitor herbicide gene comprises the following steps:
  • a plurality of microorganisms containing HPPD inhibitor herbicide-resistant microorganisms are cultured in a resistance screening medium containing an HPPD inhibitor to obtain a resistant strain, and purified and cultured to obtain a monoclonal antibody;
  • the anti-HPPD inhibitor gene can be screened by the method.
  • the species information of the HPPD inhibitor-resistant microorganism is known or unknown. If the species information is unknown, the step S12 may be increased. For details, refer to Examples 3 to 4.
  • This example is a screening step for an anti-HPPD inhibitor herbicide strain.
  • a resistance screening medium is obtained by mixing the above reagents and adding an HPPD inhibitor herbicide.
  • Mesotrione (MT) as an example, the screening medium was formulated into screening medium with different concentration gradients:
  • SMNT-MT1 1 mM mesotrione was added to SMNT medium;
  • TM5 5 mM mesotrione was added to the SMNT medium.
  • TM5 strain 2.5 ⁇ l was inoculated separately in 1 ml of TM10, TM20 and higher TM medium, and cultured at 300 r/min for 30 hours at 30 ° C to observe the growth of the bacteria. If the bacteria grew to half saturation and above, the bacteria were preserved. In summary, bacteria with different concentrations of resistance are also isolated.
  • This example is an identification step of an isolated strain.
  • the preserved strain was streaked onto LB solid medium and cultured at 30 ° C for 24 to 48 hours.
  • the 16s rRNA primer sequence is as follows:
  • 16S-27F 5-AGAGTTTGATCCTGGCTCAG-3;
  • 16S-1492R 5-GGTTACCTTGTTACGACTT-3.
  • This example is a cloning step of the HPPD gene.
  • the microorganism was judged to be MTCC5279 (Pseudomonas putida).
  • Primers were designed based on the HPPD gene sequence of the strain found in NCBI, and PCR amplification was carried out using the HPPD gene as a template.
  • the PCR product was subjected to agarose gel electrophoresis after 5 ⁇ L of the PCR product, and the remaining PCR products were sent for sequencing.
  • the open reading frame (ORF) of the HPPD gene was selected to design primers to amplify the target gene.
  • Primer design is as follows:
  • HPPD-F 5-ATGGCTGATATCTTCGACAACCCG-3
  • HPPD-R 5-TTATTCGACGTTCAGGACGCCGC-3
  • HPPD gene was cloned using the strain MTCC5279 as a template.
  • the PCR reaction system is as follows:
  • the sequencing result was the nucleotide sequence shown in SEQ ID NO: 1.
  • the PCR product was recovered using the PCR product purification kit (Omega), and the PCR was performed by restriction enzyme digestion (Pac1 and Sbf1). The product formed a cohesive end; the digested fragment was ligated with the same digested bacterial expression vector pADV3 and left overnight at 16 °C.
  • the expression vector pADV3-MT79HPPD was constructed.
  • Primer design is as follows:
  • the cleavage sites are the cleavage sites Pac1 and Sbf1, and the preceding sequence is the protection base sequence.
  • the HPPD gene was cloned using the strain MTCC5279 as a template.
  • the PCR reaction system is as follows:
  • the enzyme digestion system is as follows:
  • connection system is as follows:
  • This example is a resistance test procedure for the cloned HPPD gene.
  • the bacterial expression vector pADV3-MT79HPPD was transformed into E. coli Trans1-T1 competent cells by heat shock method, and the transformation results were verified by PCR and enzyme digestion. Due to the pADV3 vector It can be expressed in E. coli Trans1-T1 cells. Therefore, Trans1-T1 E. coli cells transformed with positive pADV3-MT79HPPD can be directly tested for resistance.
  • the monoclonal cells are activated overnight in liquid LB medium;
  • the activated bacteria were transferred into TM0, TM1, TM5, TM10 and TM20 resistance test medium, Trans1-T1 E. coli transformed with pADV3-pfHPPD was used as a positive control, Trans1-T1 transformed with empty pADV3 plasmid. E. coli was used as a negative control, and cultured at 37 ° C, 300 r / min shaker and observed changes in color.
  • the HPPD inhibitor resistance is scored according to the depth of the color of the resistance screening medium. If the color of the medium becomes dark, it means that the HPPD gene can grow in the herbicide-added medium, ie The HPPD gene is resistant to the herbicide, and the darker the color, the stronger the HPPD inhibitor resistance. If the color of the measured medium did not change, and the medium of the positive control turned dark, it indicates that the gene was not resistant to the herbicide to be measured, and the color change result of the medium of this example is shown in FIG.

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Abstract

La présente invention décrit un procédé de criblage d'un gène de résistance contre un herbicide de type inhibiteur HPPD. Le procédé comprend le criblage à haut débit d'un micro-organisme dans un milieu nutritif contenant un inhibiteur HPPD, l'utilisation du séquençage d'ARNr 16s afin d'identifier le micro-organisme criblé, l'utilisation des résultats d'identification pour déterminer les espèces et le genre du micro-organisme criblé, et le clonage d'un gène HPPD pour vérification.
PCT/CN2016/072810 2016-01-29 2016-01-29 Procédé de criblage d'un gène de résistance à un herbicide de type inhibiteur hppd et application Ceased WO2017128302A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114350689A (zh) * 2021-06-15 2022-04-15 南京厚土生物科技有限公司 一种抗性hppd基因的克隆表达的方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1192243A (zh) * 1995-06-02 1998-09-02 罗纳-普朗克农业化学公司 羟苯丙酮酸双加氧酶基因的dna序列以及含有羟苯丙酮酸双加氧酶基因的抗特定除草剂植物的获得
CN101063174A (zh) * 2007-05-28 2007-10-31 中国热带农业科学院橡胶研究所 一种规模化筛选微生物耐碱相关基因的方法
CN104736699A (zh) * 2012-09-14 2015-06-24 拜尔作物科学有限合伙公司 Hppd变体及使用方法

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1192243A (zh) * 1995-06-02 1998-09-02 罗纳-普朗克农业化学公司 羟苯丙酮酸双加氧酶基因的dna序列以及含有羟苯丙酮酸双加氧酶基因的抗特定除草剂植物的获得
CN101063174A (zh) * 2007-05-28 2007-10-31 中国热带农业科学院橡胶研究所 一种规模化筛选微生物耐碱相关基因的方法
CN104736699A (zh) * 2012-09-14 2015-06-24 拜尔作物科学有限合伙公司 Hppd变体及使用方法

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
WU, DANDAN: "Screening, Identification and Its Gene Cloning of Glyphosate Resistant Strains", SCIENCE-ENGINEERING (A), CHINA MASTER`S THESES FULL-TEXT DATABASE NO.02, 15 February 2014 (2014-02-15) *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114350689A (zh) * 2021-06-15 2022-04-15 南京厚土生物科技有限公司 一种抗性hppd基因的克隆表达的方法

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