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Tobacco chlorogenic acid synthesis gene NtHQT and application thereof

A technology for synthesizing genes and chlorogenic acid, which is applied in application, genetic engineering, plant genetic improvement, etc.

Active Publication Date: 2020-03-24
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although one HQT gene has been cloned in tobacco, whether other homologous genes of HQT also play an important role in the synthesis of chlorogenic acid still needs further experimental verification

Method used

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  • Tobacco chlorogenic acid synthesis gene NtHQT and application thereof
  • Tobacco chlorogenic acid synthesis gene NtHQT and application thereof
  • Tobacco chlorogenic acid synthesis gene NtHQT and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Tobacco NtHQT The process of gene cloning and sequence analysis is briefly described as follows.

[0050] (1) Cloning of NtHQT gene

[0051] Take the four-week-old safflower Dajinyuan leaf as a sample, refer to the kit instructions, extract its total RNA, and use DNase I to remove a small amount of DNA remaining in the RNA, and then use a spectrophotometer to determine the OD 230 , OD 260 And OD 280 Value and calculate the concentration, and use a bioanalyzer to determine the integrity of RNA;

[0052] Further refer to the instructions of the reverse transcription kit to select high purity and complete total RNA for the synthesis of cDNA strand I.

[0053] Based on NCBI database, and existing HQT Gene sequence and related BLAST analysis results, the inventor designed the primer sequence for PCR amplification as follows:

[0054] Upstream primer NtHQT-F: 5'-ATGAAAGAAGCATTAAGTAATG-3',

[0055] Downstream primer NtHQT-R: 5'-AAATTCATACAAGTACTTCTCA-3'.

[0056] Then, using the above-pr...

Embodiment 2

[0071] To confirm the cloned NtHQT Gene function. For this gene, the inventors further prepared an overexpression vector to lay a foundation for the construction of related transgenic plants and the verification of gene function. This example briefly describes the construction process of related overexpression vectors as follows.

[0072] First, for the target gene NtHQT obtained in Example 1, primers for PCR amplification (the purpose is to increase the restriction sites of BsmBI and Esp3II) are designed as follows:

[0073] Upstream primer F: 5'-CAGTCGTCTCACAACATGAAAGAAGCATTAAGTAA-3',

[0074] Downstream primer R: 5'-CAGTCGTCTCATACAAAATTCATACAAATACTTCT-3';

[0075] Then, the recombinant pMD19-T plasmid vector containing the NtHQT gene prepared in Example 1 was used as a template to perform PCR amplification, and the PCR amplification product was recovered and purified.

[0076] The PCR amplified products were double digested with BsmBI and Esp3II, and the pBWA(V)HS-GLosgfp vector was ...

Embodiment 3

[0080] Based on the overexpression vector prepared in Example 2, the inventors further transformed it into tobacco plants, screened and cultivated to obtain new transgenic tobacco varieties. The specific process is briefly described as follows.

[0081] (1) Preparation of Agrobacterium competent cells

[0082] Pick a single colony of Agrobacterium GV3101 in 2ml of LB (containing 20 mg / mL Rif) and culture overnight at 28°C, then take 2 ml of well-growing bacterial solution (containing 25 mg / L Rif) and inoculate it in 50 ml of LB liquid medium Medium, shake culture at 28℃ to OD 600 = About 0.5;

[0083] Transfer the bacterial solution to a 50 ml centrifuge tube, place it on ice for 30 minutes, and then collect the bacteria by centrifugation (4°C, 5000 rpm for 5 minutes);

[0084] Lightly suspend the bacteria in 10 ml of 0.15 M pre-cooled sodium chloride solution, and collect the bacteria by centrifugation (4°C, 5000 rpm for 5 minutes);

[0085] Discard the supernatant, add 20 ml of pre-c...

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Abstract

The invention belongs to the technical field of plant genetic engineering, and particularly relates to a tobacco chlorogenic acid synthesis gene NtHQT and an application thereof. The full length of the gene is 4086 bp. The gene comprises an intron and two exons, and the length of a coding region is 1128 bp. Transferase encoded by the tobacco chlorogenic acid synthesis gene NtHQT contains 375 aminoacids. On the basis of the existing tobacco genetic engineering, an inventor clones to obtain a new tobacco HQT gene (NtHQT) related to the content of chlorogenic acid. In the further functional verification process, the inventor performs overexpression on the gene, and the result shows that after the gene is overexpressed, the chlorogenic acid content in a new transgenic plant is obviously improved. The result further proves that the gene is indeed related to the content of chlorogenic acid in tobacco leaves. Based on the result, a certain application foundation and reference can be laid forquality regulation and new tobacco variety cultivation in the tobacco growth process.

Description

Technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a tobacco chlorogenic acid synthesis gene NtHQT and its application patent application. Background technique [0002] Chlorogenic acid (CGA), also known as coffee tannin, is a kind of phenylpropanoids produced by plants through the phenylalanine pathway during aerobic respiration. Chlorogenic acid is widely present in coffee beans, tea, fruits, vegetables and various grains, as well as in various Chinese medicinal materials. Studies have shown that chlorogenic acid has broad-spectrum anti-inflammatory, anti-bacterial, anti-viral, anti-mutation, prevention of hypertension, coronary heart disease and other biomedical functions. [0003] Studies on the physiological metabolism of chlorogenic acid have shown that multiple enzymes of the benzoic acid metabolic pathway are involved in the synthesis of chlorogenic acid. Among them, hydroxycinnamoyl-CoA quinatehyd...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/82A01H5/12A01H6/82
CPCC12N9/1029C12N15/825C12N15/8218C12Y203/01099
Inventor 武明珠王中谢小东张剑锋魏攀李锋罗朝鹏杨军
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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