Aluminum-resistant related gene GmNAC069 and encoding protein and application thereof
A protein and encoding technology, applied in the field of aluminum resistance related gene GmNAC069 and its encoded protein and application
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Embodiment 1
[0051] Example 1. Cloning and expression of soybean aluminum tolerance-related gene GmNAC069
[0052] 1. Cloning of Soybean Aluminum Tolerance Related Gene GmNAC069
[0053] The root total RNA of soybean material Huaxia No. 3 (aluminum-tolerant soybean variety) was extracted with a plant total RNA extraction kit (TR02, GeneMark), and its integrity was detected by 1% agarose electrophoresis.
[0054] The total RNA was reverse-transcribed to obtain cDNA, and the cDNA was synthesized according to the instructions of the PrimeScriptTMRT reagent Kit with gDNA Eraser kit.
[0055] Design the following primers:
[0056] Sequence 3: 5'-ATGAAGGGAGAATTAGAGTTG-3'
[0057] Sequence 4: 5'-TCACATCTTCTGTAGGTACATG-3'
[0058] Using the above cDNA as a template, PCR amplification is performed with the primers shown in sequence 3 and sequence 4 to obtain a PCR amplification product.
[0059] The above PCR amplification system is a PCR reaction solution (50 μl system) configured in the follo...
Embodiment 2
[0083] Example 2, the acquisition of transgenic GmNAC069 soybean
[0084] 1. Transgenic GmNAC069 soybeans
[0085] 1. Obtaining the recombinant vector
[0086] Recombinant plant expression vector pTF101.1-GmNAC069 (such as figure 1Shown) is the plasmid obtained by replacing the fragment between the Xba I and Sac I restriction sites of the pTF101.1 vector with the GmNAC069 gene shown in sequence 1, and the full-length sequence of GmNAC069 is inserted forward into the CaMV35S promoter.
[0087] 2. Recombinant bacteria
[0088] The recombinant vector pTF101.1-GmNAC069 was transformed into Agrobacterium EHA105 (BioVector NTCC Inc.) with a Bio-rad electric shock transformation instrument to obtain recombinant Agrobacterium EHA105 / pTF101.1-GmNAC069 (using the primers shown in sequence 5 and sequence 6 For PCR amplification of bacterial solution, 180bp is positive).
[0089] 3. Transgenic GmNAC069 soybean
[0090] Soybean was transformed by Agrobacterium EHA105-mediated transfor...
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