Gene for regulating flavonoid synthesis and encoded protein and application of gene

A flavonoid, gene technology, applied in application, genetic engineering, plant genetic improvement and other directions

Active Publication Date: 2020-02-07
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, whether this transcription factor is involved in the regulation of flavonoid synthesis has not been reported

Method used

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  • Gene for regulating flavonoid synthesis and encoded protein and application of gene
  • Gene for regulating flavonoid synthesis and encoded protein and application of gene
  • Gene for regulating flavonoid synthesis and encoded protein and application of gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] The cloning of tobacco NtMYB59 gene sequence comprises the following steps:

[0035] (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, whic...

Embodiment 2

[0047] The construction of tobacco NtMYB59 gene expression vector comprises the following steps:

[0048] (1) Sequence the correct plasmid and pCAMBIA1301 plasmid using XbaI and KpnI double enzyme digestion, digest at 37°C for 2 hours, and then undergo agarose gel electrophoresis to recover the target gene fragment and the cut pCAMBIA1301 plasmid, and configure the connection in a microcentrifuge tube System: digested pCAMBIA1301 plasmid 1μl, target gene fragment 5μl, T4buffer 1μl, T4 ligase 1μl, dd-H 2 O2 μl. Ligation reaction was carried out overnight at 16°C, transformed into Escherichia coli competent cells, and positive clones were selected 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 380bp in the coding region of the NtMYB59 gene, and then reversely ...

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Abstract

The invention relates to a gene for regulating flavonoid synthesis and an encoded protein and application of the gene, and belongs to the technical field of plant gene engineering. A gene sequence ofthe gene for regulating flavonoid synthesis is as shown in SEQ ID NO: 1. The gene derives from common tobacco, is named NtMYB59, the full length of the NtMYB59 gene is 9552bp, the NtMYB59 gene includes 10 introns and 11 exons, and the total length of a coding region of the NtMYB59 gene is 1251bp. According to the gene for regulating flavonoid synthesis and the encoded protein and application of the gene, through technologies of cloning and analysis of gene sequences, construction of expression vectors, gene expression detection and the like, it is found that NtMYB59 transcription factors can inhibit the synthesis of flavonoids in leaves, and the inhibition is achieved by inhibiting the expression of NtFLS and NtMYB12 genes; and by changing the expression level of NtMYB59 gene, the contentof the flavonoids in plant leaves can be significantly changed, and a theoretical basis and target genes for improving crop quality are provided.

Description

technical field [0001] The invention relates to a gene for regulating flavonoid synthesis, its encoded protein and its application, and belongs to the technical field of plant genetic engineering. Background technique [0002] Flavonoids are a class of secondary metabolites of small molecules that can affect the life activities of plants, animals and microorganisms at different subcellular sites. In plants, flavonoids are involved in inhibiting the polar transport of auxin, resisting biotic and abiotic stress, allelopathy, regulating the level of active oxygen in plants, attracting insects to help pollination, promoting pollen germination and pollen tube elongation, absorbing ultraviolet light and other physiological processes. In animals, flavonoids also have various physiological activities, such as anti-oxidation, regulating angiogenesis, interacting with protein kinases, and causing apoptosis. [0003] Flavonoids can be divided into several categories according to the ...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C12N15/66C12N5/10C07K14/415
CPCC07K14/415C12N15/8243
Inventor 王中杨军王姗姗武明珠魏攀王燃罗朝鹏张剑锋李泽锋李锋
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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