A gene regulating flavonoid synthesis and its encoded protein and application

A gene and protein technology, applied in the field of plant genetic engineering, can solve the problem of not detecting flavonols in genetically modified materials, and achieve the effect of increasing the content

Active Publication Date: 2021-07-02
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, they did not detect the content of flavonols in the transgenic materials, so whether this transcription factor regulates the synthesis of flavonols has not been reported yet

Method used

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  • A gene regulating flavonoid synthesis and its encoded protein and application
  • A gene regulating flavonoid synthesis and its encoded protein and application
  • A gene regulating flavonoid synthesis and its encoded protein and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The cloning of tobacco NtMYB27 gene sequence comprises the following steps:

[0037] (1) Tobacco seedling leaf tissues were immediately frozen with liquid nitrogen after harvesting, and then stored in a -80°C refrigerator. After being fully ground with liquid nitrogen, the tobacco RNA rapid extraction kit (Gene Answer, RT0110) was used for extraction, and the reagents were strictly followed. The box instructions were used to extract total RNA from tobacco leaves. During the extraction process, DNase I (Omega, #E1091-01) was used to digest and remove genomic DNA. After the extraction was completed, the quality of RNA was detected by agarose gel, and the concentration of RNA samples was determined by Nanodrop. Select samples with good quality and concentration to synthesize the first strand of cDNA. The synthesis process uses PrimeScript TM II 1stStrand cDNA Synthesis Kit (TAKARA, 6210A), in strict accordance with the instructions of the kit, to obtain cDNA samples, which...

Embodiment 2

[0047] The construction of tobacco NtMYB27 gene expression vector comprises the following steps:

[0048] (1) Sequence the correct plasmid and pCAMBIA1301 plasmid using XbaⅠ and KpnⅠ double restriction digestion, and the schematic diagram of the pCAMBIA1301 plasmid structure is as follows Figure 4 shown. After digestion at 37°C for 2 hours, the target gene fragment and the cut pCAMBIA1301 plasmid were recovered by agarose gel electrophoresis. Configure the ligation system in a microcentrifuge tube: digested pCAMBIA1301 plasmid 1μl, target gene fragment 5μl, T4buffer 1μl, T4 ligase 1μl, dd-H 2 O 2 μl, 16 ℃ ligation reaction overnight, transform Escherichia coli competent cells, select positive clones to extract plasmids.

[0049] (2) After the plasmid is identified by PCR, it is transformed into competent Agrobacterium and used for transgenic tobacco. The construction of the antisense expression vector is to use the following primers to amplify a fragment of about 250bp in t...

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Abstract

The invention relates to a gene for regulating the synthesis of flavonoids, its coded protein and its application, and belongs to the technical field of plant genetic engineering. The gene regulating flavonoid synthesis, the sequence is shown in SEQ ID NO: 1, which is derived from common tobacco, named NtMYB27, the NtMYB27 gene is 1240bp in length, contains 3 introns and 4 exons, and its coding region is complete 597 bp long. The present invention finds that the NtMYB27 transcription factor can inhibit the synthesis of flavonols in leaves through cloning analysis of gene sequences, construction of expression vectors, and gene expression detection, and this inhibition is achieved by inhibiting the expression of NtFLS and NtCHS genes of. Therefore, the present invention confirms that NtMYB27 is a negative regulator of plant flavonol synthesis. When the expression of this gene is inhibited or its function is lost, it can significantly increase the content of flavonols in plant leaves, providing a basis for the subsequent use of gene editing technology to enhance plant flavonols. Assays provide clear target genes.

Description

technical field [0001] The invention relates to a gene for regulating the synthesis of flavonoids, its coded protein and its application, and belongs to the technical field of plant genetic engineering. Background technique [0002] Tobacco is one of the main economic crops and occupies an important position in our national economy. The polyphenols in tobacco mainly include tannins, coumarins and flavonoids. The active ingredients of the flavonoid flavoring beads enter the oral cavity of the human body along with the smoke in the cigarette, and can directly act on the throat, playing a good role in improving the comfort of the throat. [0003] The content of flavonoids in plants is affected by light intensity, ultraviolet rays, temperature, nitrogen levels, bacterial infection, drought and so on. In addition, plant hormones can also regulate the synthesis of flavonoids, such as abscisic acid (ABA), jasmonate acid (JA) and cytokinins (cytokinins) can induce the synthesis of...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/00A01H6/82
CPCC07K14/415C12N15/8243
Inventor 王中杨军王姗姗武明珠张剑锋李锋谢小东
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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