A transcription factor avbhlh3 involved in the synthesis and regulation of bornyl acetate in A. chinensis and its application
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
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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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