Transcription factor AvbHLH3 participating in regulation and control of synthesis of bornyl acetate in amomum villosum and application of transcription factor AvbHLH3

A technology based on bornyl acetate and transcription factors, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve problems such as unclear transcription factors and unidentified transcription factors

Active Publication Date: 2021-09-21
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Up to now, the upstream transcription factors of bornyl acetate biosynthesis in Yangchunsha are not clear, and the transcription factors that regulate the biosynthesis of bornyl acetate have not been identified

Method used

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  • Transcription factor AvbHLH3 participating in regulation and control of synthesis of bornyl acetate in amomum villosum and application of transcription factor AvbHLH3
  • Transcription factor AvbHLH3 participating in regulation and control of synthesis of bornyl acetate in amomum villosum and application of transcription factor AvbHLH3
  • Transcription factor AvbHLH3 participating in regulation and control of synthesis of bornyl acetate in amomum villosum and application of transcription factor AvbHLH3

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1 Cloning of the transcription factor AvbHLH3 gene of Yangchunsha and its bioinformatics analysis

[0028] (1) Extraction of total RNA and synthesis of the first strand of cDNA from the fruit of Yangchunsha

[0029] Take freshly harvested 100mg of Cinnamomum indica fruit, grind it into powder under liquid nitrogen. Polysaccharide polyphenol plant RNA extraction kit (Huayueyang Biotechnology Co., Ltd., item number: 0416-50) was used to extract total RNA from the fruit of Amaryllis japonica. Using NanoDrop TM 2000c ultra-micro spectrophotometer (Thermo Scientific, Wisconsin, USA) and 1.0% agarose gel electrophoresis (Biorad, California, USA) were used to determine the content and purity of total RNA. Take 2 μg of the purified total RNA of Cinnamomum mellifera fruit, and synthesize the first strand of cDNA according to the instruction manual of the reverse transcriptase M-MLV kit (Promega Company, product number: M1701), and dilute the reaction product to the des...

Embodiment 2

[0035] Example 2 Analysis of gene expression pattern of AvbHLH3 and AvBPPS in Yangchunsha

[0036] Collect different tissues (roots, stems, leaves, flowers, and fruits) of C. chinensis, and perform total RNA extraction and reverse transcription reaction according to the method of Example 1-(1). The real-time fluorescent quantitative PCR reaction designed the upstream primer (SEQ ID NO.5) and the downstream primer (SEQ ID NO.6) respectively according to the AvbHLH3 gene, respectively designed the upstream primer (SEQ ID NO.7) and the downstream primer (SEQ ID NO.6) according to the AvBPPS gene. .8), using SYBR Premix Ex Taq TM Kit (Takara, Cat. No.: DRR420A) was used for real-time fluorescent quantitative PCR amplification. The reaction program was denaturation at 95°C for 2 min, followed by 40 cycles of reaction (95°C for 15 s, 60°C for 1 min). react in 480Instrument real-time fluorescent quantitative PCR (Roche Diagnostics, Mannheim, Germany) obtained data after running,...

Embodiment 3

[0038] Content analysis of bornyl acetate content in the volatile oil of C.

[0039] The determination of bornyl acetate content in the volatile oil of C. chinensis is carried out according to the item of Amomum chinensis in the Pharmacopoeia of the People's Republic of China. 200 mg of C. chinensis roots, stems, leaves, flowers and fruits are ground into powder under liquid nitrogen, and 1.5 mL of n-hexane is added. , sonicated in an ice-water bath for 30 min, and then placed in a 55°C water bath for 1 h. Centrifuge at 10000×g for 10 min, and transfer the supernatant to a 1.5 mL sample bottle for detection.

[0040] The gas chromatography-mass spectrometry detector (GC-MS, QP2010SE, Shimadzu Corporation, Japan) equipped with HP5-MS quartz capillary column (30m×0.25mm×0.25μm, Supelco, USA) was used for determination and analysis. Use helium as the carrier gas at a flow rate of 1 mL min -1 . The inlet temperature was 230°C, and a 10:1 split mode was used. The GC temperature...

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Abstract

The invention discloses a transcription factor AvbHLH3 participating in regulation and control of synthesis of bornyl acetate in amomum villosum and application of the transcription factor AvbHLH3. The invention provides the transcription factor AvbHLH3 participating in regulation and control of synthesis of bornyl acetate in amomum villosum, and the nucleotide sequence of the transcription factor AvbHLH3 is as shown in SEQ ID NO. 1. In the invention, the Amomum villosum transcription factor AvbHLH3 can be combined with a terpenoid synthase gene AvBPPS promoter region G-box (CACGTG) site, and the expression of the AvBPPS gene is activated, so that the biosynthesis of bornyl acetate in Amomum villosum is promoted. The content of bornyl acetate in the amomum villosum volatile oil can be remarkably increased by over-expressing the amomum villosum transcription factor AvbHLH3 gene by adopting a genetic transformation technology. Therefore, the AvbHLH3 can obviously promote the biosynthesis of bornyl acetate in the amomum villosum volatile oil and improve the medicinal quality of the amomum villosum volatile oil.

Description

Technical field: [0001] The invention belongs to the technical field of genetic engineering of medicinal plants, and specifically relates to a transcription factor AvbHLH3 involved in the biosynthesis and regulation of bornyl acetate of Dipinscus chinensis and its related materials and applications. Background technique: [0002] Amomum villosum is a medicinal plant of the Chinese herbal medicine Amomum villosum, which is produced in Yangchun City, Guangdong Province. Yangchunsha is rich in terpenoid volatile oils, especially bornyl acetate, borneol and camphor, which are included in various editions of the Pharmacopoeia of the People's Republic of China, and bornyl acetate is used as Amomum quality evaluation standard. Modern medical research shows that bornyl acetate has antidiarrheal, analgesic, anti-oxidative, anti-inflammatory and anti-cancer effects (Li Xiaoguang, Ye Fuqiang, Xu Honghua. Study on the pharmacological effects of bornyl acetate in Amomum volatile oil[J]....

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/84A01H5/00A01H6/00
CPCC07K14/415C12N15/8247
Inventor 俞振明段俊何春梅
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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