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Tobacco bhlh transcription factor gene ntfama and its application
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A transcription factor and tobacco technology, applied in the field of tobacco genetic engineering, can solve the problems of low phenol content tobacco production, reduction of phenol precursors, etc.
Active Publication Date: 2022-07-29
CHINA TOBACCO YUNNAN IND
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Therefore, the regulation of phenylpropane substances can regulate CGA. Studies have found that many transcription factors can regulate phenylpropane substances by binding to the promoters of key synthetase genes in the phenylpropane pathway (Mu Hongmei et al., 2015) [4] , expressing snapdragon in tomato wxya gene increases anthocyanin accumulation, and expression of this gene in tobacco also induces anthocyaningene expression, resulting in increased anthocyanin content (Mooney et al., 1995) [5] However, the research on the effect of bHLH on the regulation of chlorogenic acid substances has not been reported. In our previous research, we found a significant correlation between a bHLH transcription factor gene and the content of chlorogenic acid. By regulating the expression of this gene, the effective regulation of tobacco CGA content can be achieved. , can realize the control of tobacco CGA content from the source, and then achieve the goal of reducing phenol precursors, and provide a reference and basis for the production of low phenol content tobacco leaves
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Embodiment 1
[0039] Tobacco in this example NtFAMA The construction process of gene cloning and silencing vector is briefly introduced as follows.
[0041] According to previous studies on the tobacco genome and related NtFAMA For gene research, select the specific coding sequence as the target fragment, and design the primer sequences for PCR amplification as follows:
[0042] NtFAMA-F: 5'-TTTGCCTACAATTTCCCCAA-3',
[0043] NtFAMA-R: 5'-CTTGGCATGAGAGACCTCAA-3';
[0044] Using the cDNA of tobacco K326 leaves as the template, PCR amplification was carried out to obtain NtFAMA Gene;
[0045] The PCR amplification program was as follows: 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;
[0056] On the basis of Example 1, using the VIGS technology mediated by Agrobacterium, the inventor further transformed the constructed recombinant TRV2-NtFAMA vector into tobacco plants, and verified and analyzed the phenotypic changes of the relevant plants. The specific experimental process was introduced. as follows.
[0058] 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:
[0059] The positive cloned plasmids of TRV2-GFP (vector control), TRV2-PDS (VIGS efficiency control) and TRV2-NtFAMA were transformed into Agrobacterium GV3101 competent cells by electroporation, respectively. The YEB plates of mg / L Rif were cultured and screened, and after 2 d inversion culture at 28°C, Agrobacterium with the target gene ...
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Abstract
The invention belongs to the field of tobacco genetic engineering, in particular to tobacco transcription factor genes NtFAMA and its application patent application. The base sequence of the gene is shown in SEQ ID NO.1. Tobacco transcription factorgene NtFAMA It consists of 287 amino acid residues, of which the amino acidtransporter domain at the 91-142nd amino acid is conserved. This protein is related to the content of chlorogenic acid in plant leaves. After reducing the expression of this protein, the content of chlorogenic acid in leaves is significantly increased. The present invention is directed to specific tobacco transcription factors by NtFAMA Preliminary research on , found that it was highly correlated with the content of chlorogenic acid in tobacco, and after the gene was silenced, the content of chlorogenic acid in tobacco was significantly increased. Based on this characteristic, it can provide a certain application basis and reference for the cultivation of new tobacco varieties with reasonable chlorogenic acid content.
Description
technical field [0001] The invention belongs to the field of tobacco genetic engineering, in particular to tobacco transcription factor genes NtFAMA and its applications. 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] . Phenol also has toxic effects such as mutagenicity and genotoxicity, and is included in the EPA list of harmful ingredients (US.EPA, 2002). [0003] Foreign researchers have proved that polyphenolic compounds (chlorogenic acid: chlorogenic acid, CGA and rutin) have better ef...
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