A regulatory gene that reduces the total protein and phenol content of tobacco leaves
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A technology for regulating gene and protein content, applied in the field of tobacco genetic engineering
Active Publication Date: 2022-07-29
CHINA TOBACCO YUNNAN IND
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but Ntε-LCY2 There is no relevant report on the research of genes on phenol
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Embodiment 1
[0035] Tobacco in this example Ntε-LCY2 The construction process of gene cloning and silencing vector is briefly introduced as follows.
[0036] (1) Tobacco Ntε-LCY2 gene cloning
[0037] According to previous studies on the tobacco genome and related Ntε-LCY2 For gene research, select the specific coding sequence as the target fragment, and design the primer sequences for PCR amplification as follows:
[0038] Ntε-LCY2-F: 5′-TCTTAGGTGTGTGGAGGCAG-3′,
[0039] Ntε-LCY2-R: 5'-AGAATTTCCCTGAGGCAGCA-3'.
[0040] Using the cDNA of tobacco K326 leaves as the template, PCR amplification was carried out to obtain Ntε-LCY2 Gene;
[0041] The PCR amplification program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15s, annealing at 55°C for 15s, extension at 72°C for 30s, and after 34 cycles, complete extension at 72°C for 5min;
[0042] The PCR amplification products were detected by agarose gel electrophoresis, and the electrophoresis products were ...
Embodiment 2
[0054] On the basis of Example 1, using the VIGS technology mediated by Agrobacterium, the inventor further transformed the constructed recombinant TRV2-Ntε-LCY2 vector into tobacco plants, and made a verification analysis on the phenotypic changes of relevant plants. A brief introduction to the process is as follows.
[0055] (1) Transformation of Agrobacterium
[0056] It should be noted that, with reference to the operation of Example 1 and the prior art, the inventors simultaneously prepared TRV2-GFP and TRV2-PDS recombinant vectors as transgenic positive and negative controls, and the specific transformation process is as follows:
[0057] The positive cloned plasmids of TRV2-GFP (vector control), TRV2-PDS (VIGS efficiency control) and TRV2-Ntε-LCY2 were transformed into Agrobacterium GV3101 competent cells by electroporation, respectively. 50mg / L Rif YEB plate was cultured and screened. After 2d inversion culture at 28°C, colony PCR was used to screen Agrobacterium with...
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Abstract
The invention belongs to the field of tobacco genetic engineering, and in particular relates to a regulation gene tobacco lycopene ε-cyclase gene for reducing the total protein and phenol content of tobacco leaves Ntε‑LCY2, The base sequence of the gene is shown in SEQ ID NO.1. Tobacco lycopene ε-cyclase Ntε-LCY2 consists of 498 amino acid residues, of which amino acids 105-287 and 290-475 are conserved transporter domains. This protein is related to the total protein content in plant leaves. After reducing the expression of this gene, the total protein content in leaves is significantly reduced. The present invention makes use of specific Ntε‑LCY2 Preliminary study of , found that it was highly correlated with the total protein content of tobacco, and the total protein content of tobacco was significantly reduced after the gene mutation. Based on this characteristic, it can provide a certain application basis and reference for the cultivation of new tobacco varieties with low phenol content.
Description
technical field [0001] The invention belongs to the field of tobacco genetic engineering, in particular to tobacco lycopene ε-cyclase gene Ntε-LCY2 application. Background technique [0002] Phenol is listed as one of the seven representative harmful components under key control by the State Tobacco Monopoly Administration. It is rapidly distributed into all tissues after inhalation with smoke, and has significant mucosal permeability, leading to tissue necrosis and corrosion and shedding. Long-term inhalation of phenol-containing gases can lead to upper airway wheezing, respiratory distress and even failure, as well as headaches, dizziness, kidney damage and heart disease, and cancer. [1-7] . Phenol also has toxic effects such as mutagenicity and genotoxicity, and is included in the EPA list of harmful ingredients (US.EPA, 2002). [0003] The sources and precursors of phenol in smoke have been clarified. Therefore, the application of biotechnology to interfere with the c...
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