Herbicide resistance protein and encoding genes thereof and application thereof
A resistance gene and herbicide technology, applied in genetic engineering, plant gene improvement, application, etc., can solve the problem of undiscovered 24DT02 herbicide resistance protein expression level, herbicide tolerance report, etc., to improve flexibility, The effect of high flexibility
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no. 1 example 、24D
[0098] The first embodiment, the acquisition and synthesis of 24DT02 gene sequence
[0099] 1. Obtain the 24DT02 gene sequence
[0100] The amino acid sequence (298 amino acids) of the 24DT02 herbicide resistance protein is shown in SEQ ID NO: 2 in the sequence listing; the amino acid sequence (298 amino acids) encoding the 24DT02 herbicide resistance protein corresponding to said 24DT02 herbicide resistance protein is obtained according to the plant-preferred codon amino acids) nucleotide sequence (897 nucleotides), as shown in SEQ ID NO:1 in the sequence listing.
[0101] 2. Synthesize the above-mentioned 24DT02 nucleotide sequence
[0102] The 24DT02 nucleotide sequence (shown as SEQ ID NO: 1 in the sequence listing) was synthesized by Nanjing KingScript Biotechnology Co., Ltd.; the 5' of the synthesized 24DT02 nucleotide sequence (SEQ ID NO: 1) A SpeI restriction site is also connected to the end, and a KasI restriction site is also connected to the 3' end of the 24DT02 ...
no. 2 example
[0106] The second embodiment, construction of Arabidopsis recombinant expression vector and transformation of recombinant expression vector into Agrobacterium
[0107] 1. Construction of Arabidopsis thaliana recombinant cloning vector DBN01-T containing 24DT02 nucleotide sequence
[0108] The synthetic 24DT02 nucleotide sequence was ligated into the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the operation steps were carried out according to the instructions of the pGEM-T vector produced by Promega Company to obtain the recombinant cloning vector DBN01-T, which Build process such as figure 1 Shown (wherein, Amp represents the ampicillin resistance gene; f1 represents the replication origin of phage f1; LacZ is the LacZ initiation codon; SP6 is the SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter; 24DT02 is 24DT02 Nucleotide sequence (SEQ ID NO: 1); MCS is multiple cloning site).
[0109] Then, the recombinant cloning vector DBN01-T was tra...
no. 3 example
[0125] The third embodiment, the acquisition of Arabidopsis plants transferred to 24DT02 nucleotide sequence
[0126] Wild-type Arabidopsis seeds were suspended in 0.1% agarose solution. Suspended seeds were stored at 4°C for 2 days to fulfill the need for dormancy to ensure simultaneous seed germination. Mix horse manure with vermiculite and subirrigate with water until moist, allowing the soil mixture to drain for 24 hours. The pretreated seeds were planted on the soil mixture and covered with a moisture hood for 7 days. Let the seeds germinate and keep the light intensity at 120-150μmol / m at constant temperature (22°C) and constant humidity (40-50%) 2 Plants were grown in the greenhouse under long-day conditions (16 h light / 8 h dark) of 2 sec. Start by watering the plants with Hoagland's nutrient solution, followed by deionized water, keeping the soil moist but not soggy.
[0127] Transform Arabidopsis using the flower soak method. Inoculate one or more 15-30ml precult...
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