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Regulatory gene for reducing total protein of tobacco leaves and phenol content in smoke
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A technique for regulating genes and total proteins, applied in the field of tobacco genetic engineering
Active Publication Date: 2021-10-26
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] In this example, tobacco Ntε-LCY2 The process of gene cloning and silencing vector construction is briefly introduced as follows.
[0037] According to previous studies on the tobacco genome and related Ntε-LCY2 Gene research, select the specific coding sequence as the target fragment, and design the primer sequence 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 a template, PCR amplification was carried out to obtain Ntε-LCY2 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;
[0054] On the basis of Example 1, using the VIGS technology mediated by Agrobacterium, the inventors further transformed the constructed recombinant TRV2-Ntε-LCY2 vector into tobacco plants, and verified and analyzed the phenotypic changes of related plants. Specific experiments A brief introduction to the process is as follows.
[0056] It should be noted that, referring to the operation of Example 1 and the prior art, the inventor simultaneously prepared TRV2-GFP and TRV2-PDS recombinant vectors as positive and negative controls for transgenes. The specific transformation process is as follows:
[0057] The positive cloning plasmids of TRV2-GFP (vector control), TRV2-PDS (VIGS efficiency control) and TRV2-Ntε-LCY2 were transformed into Agrobacterium GV3101 competent cells by electric shock transformation respectively, using 50 mg / L Kan and The YEB plates with 50 mg / L Rif were cultured and screened, and after inverted culture at 2...
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Abstract
The invention belongs to the field of tobacco geneengineering, and particularly relates to a regulatory gene tobacco lycopene epsilon-cyclasegene Nt epsilon-LCY2 for reducing total protein of tobacco leaves and phenol content in smoke. The base sequence of the gene is shown as SEQ ID NO.1. The tobacco lycopene epsilon-cyclase Nt epsilon-LCY2 is composed of 498 amino acid residues, wherein the 105 to 287 amino acid and the 290 to 475 amino acid are conservative transport protein structural domains. The protein is related to the total protein content in plant leaves, and after the gene expression is reduced, the total protein content in the leaves is obviously reduced. Preliminary research on the specific Nt epsilon-LCY2 shows that the specific Nt epsilon-LCY2 is highly related to the total protein content of the tobacco, and after the gene is mutated, the total protein content of the tobacco is obviously reduced. On the basis of the characteristic, a certain application basis and reference can be provided for the cultivation of new varieties of low-phenol-content tobaccos.
Description
technical field [0001] The invention belongs to the field of tobacco genetic engineering, in particular to tobacco lycopene epsilon-cyclase gene Ntε-LCY2 application. Background technique [0002] Phenol is listed by the State Tobacco Monopoly Administration as one of the seven representative harmful components under key control. It will quickly distribute into all tissues after being inhaled with smoke, and has significant mucosal permeability, leading to tissue necrosis and corrosion. Long-term inhalation of phenol-containing gas can lead to upper respiratory tract wheezing, respiratory distress and even failure, as well as headaches, dizziness, kidney damage, heart disease, and cancer [1-7] . Phenol also has toxic effects such as mutagenicity and genotoxicity, and is included in the list of EPA hazardous ingredients (US.EPA, 2002). [0003] The sources and precursors of phenol in smoke have been clarified. Therefore, the application of biotechnology to interfere with t...
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