Kale 2-oxygen-dependent dioxygenase gene ba2odd1 and its application
A dioxygenase-dependent technology, applied in applications, genetic engineering, oxidoreductase, etc.
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Embodiment 1
[0031] Example 1 Cloning of kale 2-oxygen-dependent dioxygenase gene Ba2ODD1
[0032] 1. Method
[0033] 1. Extraction of kale DNA and RNA
[0034] The improved CTAB method (Murry&Thomas, 1980) was used to extract the total DNA from the kale experimental material; the RNA extraction was carried out with reference to the extraction kit of Magen Company (Catalog No. R4151-02), and reversed into cDNA;
[0035] 2. Using the cDNA of kale banneng mushroom as a template, use the polymerase chain reaction (PCR) to amplify, and the PCR amplification primers are shown in SEQ ID NO.3-4:
[0036] Ba2ODD1-F: 5'-ACTAAAAAAAAGGTTGGAGTCCAAGTGTAC-3' (SEQ ID NO.3);
[0037] Ba2ODD1-R: 5'-GGAAACACAAAGGAAACAAC-3' (SEQ ID NO. 4).
[0038] The PCR reaction system is: 2×PCR Mix: 10 μL, upstream primer (10 μM): 1 μL, downstream primer (10 μM): 1 μL, cDNA single-stranded template: 0.5 μL, double-distilled water: 7.5 μL, a total of 20 μL.
[0039]The PCR reaction program was: pre-denaturation at 95°...
Embodiment 2
[0043] Example 2 Construction of kale Ba2ODD1 gene RNAi interference vector
[0044] According to the conserved structural segment of the kale Ba2ODD1 gene sequence obtained in Example 1 and the multiple cloning site of the pFGC5941 vector, a 338bp specific fragment primer was designed, and the schematic diagram of the vector structure is as follows image 3 . Ba2ODD1 gene sense fragment amplification primer Ba2ODD1-RNAi1 and antisense fragment amplification primer Ba2ODD1-RNAi2 are shown in SEQ ID NO.5-8:
[0045] Ba2ODD1-RNAi1-F: 5'-CGGATTTAAATGCAAGATCCAGAAGCGAGGA-3' (SEQ ID NO.5);
[0046] Ba2ODD1-RNAi1-R: 5'-CCGCCATGGGCTCAGGACACCGCGGGTAA-3' (SEQ ID NO.6);
[0047] Ba2ODD1-RNAi2-F: 5'-CATGGATCCGCAAGATCCAGAAGCGAGGA-3' (SEQ ID NO.7);
[0048] Ba2ODD1-RNAi2-R: 5'-CTACCCGGGGCTCAGGACACCGCGGGTAA-3' (SEQ ID NO. 8).
[0049] The PCR reaction system was 2×PCR Mix: 15 μL, upstream primer (10 μM): 1 μL, downstream primer (10 μM): 1 μL, cDNA single-stranded template: 1 μL, double-d...
Embodiment 3
[0053] Example 3 Agrobacterium-mediated genetic transformation
[0054] 1. Method
[0055] The kale Ba2ODD1 gene RNAi interference vector successfully constructed in Example 2 was mediated by Agrobacterium, and genetically transformed into kale to obtain interference Ba2ODD1 resistant plants; the specific method is the same as the conventional Agrobacterium transformation method, including the following steps: ( 1) Disinfection and sowing of seeds; (2) Acquisition and pre-cultivation of explants; (3) Preparation of bacterial solution; (4) Dipping; (5) Antibacterial culture; (6) Primary screening culture; (7) Re-screening culture; (8) rooting culture; (9) hardening and transplanting; (10) identification of positive seedlings.
[0056] 2. Results
[0057] More than 7,000 explants were dipped, and the herbicide glufosinate-ammonium screening concentration was 10mg / L, and finally 45 resistant seedlings were obtained. According to the bar gene sequence of the herbicide glufosina...
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