Key gene GbDAL1 for inhibiting synthesis of ginkgo flavonol glycosides and application of key gene GbDAL1
A technology of flavonol glycosides and key genes, which is applied in the field of molecular biology, can solve the problem that the transcription factor that negatively regulates flavonol synthesis is poorly understood, and achieves the effect of improving the yield of flavonol glycosides
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0030] Cloning of the GbDAL1 gene
[0031] (1) Based on Ginkgo biloba genome and Ginkgo transcriptome data, a GbDAL1 gene was screened, and primers for GbDAL1 amplification were artificially designed using Primer Premier 5.0 software as follows.
[0032] Primer ID Forward Primer Reverse Primer GbDAL1 ATGGGACGAGGTCGAGTCC AACCCACCACCCCGTGTAG
[0033] (2) PCR amplification using PrimeSTAR Max (Takara, Japan), the PCR system is as follows:
[0034]
[0035] Gently mix the above mixture, centrifuge briefly at low speed and place it in an ordinary PCR reaction instrument, set the following program:
[0036]
[0037] Gel running: Take out the gene amplification product in the PCR instrument, use the electrophoresis instrument to spot an appropriate amount of the product on a 1% agarose gel for detection, take out the application imaging system for observation after about 20 minutes, and obtain the target fragment ( figure 1 a).
[0038] (3) Ligatio...
Embodiment 2
[0056] Construction of GbDAL1 gene expression vector
[0057] (1) In this experiment, TaKaRa QuickCut restriction enzyme (TaKaRa, Japan) was used to carry out enzyme digestion reaction experiment on pRI 101-AN vector (TaKaRa, Japan, which contains GFP tag) and target gene GbDAL1. The specific reaction system is as follows:
[0058]
[0059] After the solutions in the system were mixed, they were centrifuged instantaneously, incubated in a 37°C water bath for 30 minutes, and then the enzyme digestion reaction was completed. The enzyme-cut bands were observed by agarose gel electrophoresis, and then the target gene and carrier fragments were cut and recovered for subsequent use. The carrier ligation reaction.
[0060] (2) Referring to the operation manual of TaKaRa T4 DNA Ligase (TaKaRa, Japan), connect the expression vector recovered after the double enzyme digestion reaction with the target DNA fragment product. The system is as follows:
[0061] T4 DNA Ligase ...
Embodiment 3
[0067] Regulatory factors of flavonol glycosides content in Ginkgo biloba leaves
[0068] (1) Real-time quantitative PCR technology was used to artificially design the fluorescent quantitative primer pair and internal reference (Actin) primer pair of GbDAL1 using Primer Premier 5.0 software. It was detected that GbDAL1 was highly expressed in leaves and stems of Ginkgo biloba, and its expression was lower in roots, embryos and endosperms ( image 3 a); In addition, fluorescence quantitative and transcriptome data show that the expression of GbDAL1 increases with the age of Ginkgo biloba, and has a significant age effect ( image 3 b, image 3 c).
[0069]
[0070] (2) Detection of flavonol glycosides content
[0071] The content of flavonol glycosides in Ginkgo leaves of different ages (1, 3, 5, 7, 9, 10, 20, 30 years before, during and after side branches) was further detected. A 100 mg leaf sample was first frozen in liquid nitrogen, then ground into a powder and extr...
PUM
Login to View More Abstract
Description
Claims
Application Information
Login to View More