Tobacco chloride ion channel protein gene NtCLC-F and application thereof
A chloride ion channel, chloride ion content technology, applied in the chloride ion channel protein gene NtCLC-F and its application field, can solve the problem of low transportation speed of lotus
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Embodiment 1
[0035] This example briefly introduces the process of cloning the tobacco NtCLC-F gene and constructing the silencing vector as follows.
[0036] (1) Tobacco NtCLC-F gene cloning
[0037] According to the previous research on the tobacco genome and related NtCLC-F genes, the specific coding sequence was selected as the target fragment, and the primer sequences for PCR amplification were designed as follows:
[0038] NtCLC-F-F: 5'-CTGGTAGGGAGCTTCTC-3',
[0039] NtCLC-F-R: 5'-CCTCCTAAAGAGAATGG-3'.
[0040] The cDNA of tobacco K326 (tobacco variety) leaves was used as a template, and NtCLC-F-F and NtCLC-F-R were used as primers to perform PCR amplification to obtain the NtCLC-F gene.
[0041] The PCR amplification program was: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 15 s, annealing at 55°C for 15 s, extension at 72°C for 30 s, and after 34 cycles, complete extension at 72°C for 5 min.
[0042] The PCR amplification products were detected by agarose gel ele...
Embodiment 2
[0052] On the basis of Example 1, the constructed recombinant TRV2-NtCLC-F vector was further transformed into tobacco plants using Agrobacterium-mediated VIGS technology, and the phenotypic changes of related plants were verified and analyzed. The specific experimental process is as follows.
[0053] (1) Transformation of Agrobacterium
[0054] It should be noted that referring to the operation of Example 1 and the prior art, TRV2-GFP and TRV2-PDS recombinant vectors were prepared as transgenic positive and negative controls at the same time, and the specific transformation process was as follows:
[0055] The positive cloning plasmids of TRV2-GFP (vector control), TRV2-PDS (VIGS efficiency control) and TRV2-NtCLC-F were transformed into Agrobacterium GV3101 competent cells by electric shock transformation respectively, using 50mg / L Kan and The YEB plates with 50 mg / L Rif were cultured and screened, and after inverted culture at 28°C for 2 days, the Agrobacterium with the tar...
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