Tobacco 7-dehydrocholesterol reductase gene and application thereof
A technology of dehydrocholesterol and reductase, applied in the field of tobacco genetic engineering, can solve problems such as few reports
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Embodiment 1
[0033] In this example, the cloning of the tobacco NtDHCR7 gene and the construction of the silencing vector are briefly introduced as follows.
[0034] (1) Cloning of tobacco NtDHCR7 gene
[0035] According to the previous research on the tobacco genome and related NtDHCR7 gene, the specific coding sequence was selected as the target fragment, and the primer sequences for PCR amplification were designed as follows:
[0036] NtDHCR7-F: 5'-CGCACTTCAGCTTTTGTTGC-3',
[0037] NtDHCR7-R: 5'-ACCGTGATCAGTGGAAGATGG-3'.
[0038] Using the cDNA of tobacco safflower Dajinyuan leaves (extracting the genome first, then reverse-transcribing it into cDNA) as a template, PCR amplification was performed to obtain the NtDHCR7 gene;
[0039] The PCR amplification program is: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 55°C for 15 seconds, extension at 72°C for 1 minute, and after 34 cycles, complete extension at 72°C for 5 minutes;
[0040] The PC...
Embodiment 2
[0046] On the basis of Example 1, the constructed recombinant TRV2-NtDHCR7 vector was further transformed into tobacco plants using Agrobacterium-mediated VIGS technology, and a verification analysis was performed on the phenotypic changes of related plants. The specific experimental process is as follows.
[0047] (1) Transformation of Agrobacterium
[0048] It should be noted that, referring to the operation of Example 1 and the prior art, a TRV2-GFP recombinant vector was prepared as a control, and the specific transformation process was as follows:
[0049] The positive cloning plasmids of TRV2-GFP (vector control) and TRV2-NtDHCR7 were transformed into Agrobacterium GV3101 competent cells by electric shock transformation respectively, and were cultured and screened on YEB plates containing 50 mg / L Kan and 50 mg / L Rif. After upside-down culture at 28°C for 2 days, colony PCR was used to screen the Agrobacterium with the target gene.
[0050] (2) Preparation of bacteria so...
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