Herbicide tolerance protein, its encoding gene and use
A protein and tolerance technology, used in genetic engineering, plant genetic improvement, applications, etc.
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no. 1 example
[0090] The first embodiment, isolation and identification of bacterial strains
[0091] 1. Isolation of strains
[0092] The enrichment matrix of dicamba-degrading strains was taken from biochemical sludge, compost, marine tidal flats, saline soil in Xinjiang, forest soil, farmland soil, etc. around the dicamba production plant, and was separated with a medium containing a certain concentration of dicamba. Soil samples were taken in 500mg / L dicamba low-chloride basal salt medium (dipotassium hydrogen phosphate 1.3g / L, potassium dihydrogen phosphate 0.87g / L, ammonium sulfate 0.66g / L, magnesium sulfate 0.097g / L, a Manganese sulfate hydrate 0.025g / L, ferrous sulfate heptahydrate 0.005g / L and calcium sulfate 1.26mg / L, pH 7.0; solid medium plus 15g / L agar) for 5 days (temperature 30 ° C, speed 180r / min ), transferred to the same medium with a 5% inoculum size, and transferred 3 times in a row.
[0093] Use an ultraviolet scanner to detect whether the 4th generation enrichment sol...
no. 2 example
[0100] Second embodiment, isolation and sequencing of DMT66 gene
[0101] 1. Preparation of total genome DNA of strain Ndbn-20
[0102] After the bacterial strain Ndbn-20 was cultivated in large quantities, the high-purity and large-fragmented total genomic DNA of the bacterial strain Ndbn-20 was extracted by high-salt combined with CTAB method, dissolved in TE buffer (pH8.0), and placed at temperature Store at -20°C. For specific methods, refer to the "Refined Molecular Biology Experiment Guide" (edited by F. Osper et al.).
[0103] 2. Genome sequencing and result analysis
[0104] (1) DNA sample delivery and testing
[0105] Bacterial genome sequencing requires the OD value of the DNA sample to be between 1.8-2.0, and the concentration is not lower than 30ng / μL, and the fine map requires at least 30ug of sample volume. Send sufficient DNA samples that meet the requirements to Shanghai Meiji Biomedical Technology Co., Ltd. (Beijing Branch) under dry ice insulation.
[010...
no. 3 example
[0111] The third embodiment, in vitro high-level expression and functional identification of demethylase DMT50
[0112] 1. Construction of bacterial expression vectors and acquisition of recombinant microorganisms
[0113] (1) PCR amplification of DMT50 gene
[0114] Design a pair of primers:
[0115] Primer 1: 5-CGG GGTACC ATGAGGGAGGCTGAAGTGAAGTCC-3 (the underline is the KpnI restriction site), as shown in SEQ ID NO:10 in the sequence listing;
[0116] Primer 2: 5-CCC AAGCTT CTACGCCCGCGCTGCCACGC-3 (the underline is the Hind III restriction site), as shown in SEQ ID NO: 11 in the sequence listing;
[0117] Use the following PCR amplification system to amplify the DMT50 gene from the bacterial strain Ndbn-20 genomic DNA:
[0118]
[0119] The PCR reaction conditions are: denaturation at 98°C for 1 min; then enter the following cycle: denaturation at 98°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 1 min, a total of 29 cycles; finally extension at 72°C ...
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