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Tobacco flavonoid monooxygenase gene NtCYP75B2 and application thereof

A CYP75B2, flavone monooxygenase technology, applied in the field of tobacco genetic engineering, can solve the problem of reducing phenol precursors

Active Publication Date: 2021-02-23
CHINA TOBACCO YUNNAN IND
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The above-mentioned published patents all study how to obtain new tobacco varieties with high chlorogenic acid content through gene regulation, and there is no new tobacco variety with low chlorogenic acid content through gene regulation Research on new tobacco varieties can further regulate tobacco CGA content from the source, achieve the goal of reducing phenol precursors, and provide a reference and basis for the production of low-phenol content tobacco leaves

Method used

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  • Tobacco flavonoid monooxygenase gene NtCYP75B2 and application thereof
  • Tobacco flavonoid monooxygenase gene NtCYP75B2 and application thereof
  • Tobacco flavonoid monooxygenase gene NtCYP75B2 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] In this example, the cloning of the tobacco NtCYP75B2 gene and the construction of the silencing vector are briefly introduced as follows.

[0042] (1) Cloning of tobacco NtCYP75B2 gene

[0043] According to the previous research on the tobacco genome and related CYP75B2 genes, based on the NCBI database and the existing HQT gene sequences and related BLAST analysis results, the specific coding sequence was selected as the target fragment, and the primer sequences for PCR amplification were designed as follows:

[0044] NtCYP75B2-F: 5'-AATTACGTGTTCCCACCAGC-3',

[0045] NtCYP75B2-R: 5'-CTTACCCGTCAAAGGCAAGA-3';

[0046] Tobacco flavonoid monooxygenase gene NtCYP75B2 was obtained by PCR amplification using the cDNA of tobacco K326 leaves as a template; the cDNA of tobacco K326 leaves was synthesized by reverse transcription after extracting RNA from the existing plant RNA rapid extraction kit of.

[0047] The PCR amplification program was: pre-denaturation at 95°C for 3...

Embodiment 2

[0058] On the basis of Example 1, using Agrobacterium-mediated VIGS technology, the inventors further transformed the constructed recombinant TRV2-NtCYP75B2 vector into tobacco plants, and verified and analyzed the phenotypic changes of related plants. The specific experimental process The introduction is as follows.

[0059] (1) Transformation of Agrobacterium

[0060] 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:

[0061] The positive cloning plasmids of TRV2-GFP (vector control), TRV2-PDS (VIGS efficiency control) and TRV2-NtCYP75B2 were transformed into Agrobacterium GV3101 competent cells by electric shock transformation respectively, using 50mg / L Kan and 50mg / L Kan and 50mg / L The YEB plate of L Rif was cultured and screened, and after inverting at 28°C for...

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Abstract

The invention discloses a tobacco flavonoid monooxygenase gene NtCYP75B2 and an application thereof. The base sequence of the tobacco flavonoid monooxygenase gene NtCYP75B2 is shown as SEQ ID NO.1, and a specific nucleic acid fragment is a 2 to 432-site base. Through preliminary research on specific tobacco flavonoid monooxygenase CYP75B2, it is found that the specific tobacco flavonoid monooxygenase CYP75B2 is highly related to the content of chlorogenic acid in tobacco, and after the gene is silenced, the content of chlorogenic acid in tobacco is obviously reduced. Based on the characteristic, certain application basis and reference can be provided for cultivation of new varieties of tobacco leaves with low phenol content.

Description

technical field [0001] The invention relates to the technical field of tobacco genetic engineering, in particular, the invention relates to a tobacco flavone monooxygenase gene NtCYP75B2 and its 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 airway wheezing, respiratory distress, and even failure, as well as headache, dizziness, kidney damage, 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). At present, the sources and precursors of phenol in smoke have been clarified. Therefore, the application of biotec...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/53C12N15/83C12N15/11A01H5/00A01H6/82
CPCC12N9/0004C12N15/8243
Inventor 李正风
Owner CHINA TOBACCO YUNNAN IND