Herbicide resistance protein, its encoding gene and use
A resistance gene and herbicide technology, applied in genetic engineering, plant genetic improvement, application, etc., can solve the problems of reporting herbicide tolerance without finding the expression level of AADM2 herbicide resistance protein, to achieve increased flexibility, The effect of high flexibility
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no. 1 example
[0093] The first embodiment, the acquisition and synthesis of AADM2 gene sequence
[0094] 1. Obtain the AADM2 gene sequence
[0095] The amino acid sequence (292 amino acids) of the AADM2 herbicide resistance protein is shown in SEQ ID NO: 2 in the sequence listing; the amino acid sequence (292 amino acids) corresponding to the AADM2 herbicide resistance protein is obtained according to the plant preference codon ) nucleotide sequence (879 nucleotides), as shown in SEQ ID NO:1 in the sequence listing.
[0096] 2. Synthesize the above-mentioned AADM2 nucleotide sequence
[0097] The AADM2 nucleotide sequence (shown as SEQIDNO: 1 in the sequence listing) was synthesized by Nanjing KingScript Biotechnology Co., Ltd.; the 5' end of the synthesized AADM2 nucleotide sequence (SEQIDNO: 1) is also connected with A SpeI restriction site, the 3' end of the AADM2 nucleotide sequence (SEQ ID NO: 1) is also connected with a KasI restriction site.
no. 2 example
[0098] The second embodiment, construction of Arabidopsis recombinant expression vector and transformation of recombinant expression vector into Agrobacterium
[0099] 1. Construction of Arabidopsis thaliana recombinant cloning vector DBN01-T containing AADM2 nucleotide sequence
[0100] The synthetic AADM2 nucleotide sequence was connected to 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 start codon; SP6 is the promoter of SP6 RNA polymerase; T7 is the promoter of T7 RNA polymerase; AADM2 is the nucleoside of AADM2 acid sequence (SEQ ID NO: 1); MCS is a multiple cloning site).
[0101] Then, the recombinant cloning vector DBN0...
no. 3 example
[0111] The third embodiment, the acquisition of Arabidopsis plants transferred to the AADM2 nucleotide sequence
[0112] 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. Seeds were germinated and plants were grown in a greenhouse under long-day conditions (16 h light / 8 h dark) at constant temperature (22 °C) constant humidity (40-50%) and light intensity of 120-150 μmol / m2 sec. Start by watering the plants with Hoagland's nutrient solution, followed by deionized water, keeping the soil moist but not soggy.
[0113] Transform Arabidopsis using the flower soak method. Inoculate one or more 15-30ml precultures of YEP bro...
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