A functional gene chip for identifying medicinal plant species and/or screening potential medicinal ingredients and its preparation and use method
A technology for functional genes and medicinal plants, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., which can solve the problems of difficult classification and identification, low efficiency, etc.
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Embodiment 1
[0024] Preparation of functional gene probes
[0025] Find the metabolic pathways of alkaloids, flavonoids, phenylpropanoids, quinones, steroids, terpenes, etc. in the known KEGG (www.kegg.jp) database, and find the key genes corresponding to typical Chinese medicine ingredients, Combining with the Genbank database, the screening obtained distribution in 17 genera (Vinca, Ginkgo, Astragalus, Eucommia, Artemisia, Coptidis, Heliconia, Mentha, Taxus, Rauwolfia, Stevia , Glycyrrhizae, Crocus, Foxglove, Saussurea, Scutellaria, Panax) 311 sequences of 160 functional genes in 32 species, and their numbers in Genbank are as follows:
[0026] Alkaloids
[0027] AAR02419.1, AAD42938.1|AF084972_1, Q39522, Q8W013, AAK20170.1, 2FP9_A, 2FPC_A, 2FPB_A, 2FP8_A, 2FP9_B, 2FPB_B, 2FP0_B, 2FPC_B, BAB71802.1, BAC22084.1, CAB565 1, AAC99311.1, O04847, AAC49827.1, AAC49826.1, AAB97311.1, AAR09601.1, AAR02420.1, AAR09602.1, AAR09599.1, AAR09598.1, AAR09600.1, AAM97497.1, AAK1479 1, AAD42937.1|AF08...
Embodiment 2
[0052] Fluorescent labeling of periwinkle DNA samples
[0053] As described in Example 1, mix 1.500 μg of periwinkle (Catharanthus roseus) sample DNA with 1.5 μg of hexamer base random primers to form a 25 μL system, denature at 95°C for 2 minutes, and quickly cool on ice to obtain the denatured DNA solution Mix with 15 μL of labeling reaction solution. The components of the labeling reaction solution are as follows: 5mmol / L dATP, dTTP, dGTP and 2.5mmol / LdCTP; 1mmol / L Cy5-dCTP (fluorescence red, excitation wavelength 647nm; emission wavelength 660-705nm); 2.5mmol / L dithiothreose Alcohol (DTT); 10 U Klenow Large Fragment DNA Polymerase I (Invitrogen, Calsbad, CA). The reaction mixture (40 μL) was incubated at 37°C for 2h, denatured on a heating plate at 100°C for 3min, and cooled rapidly on ice. The labeled DNA was then purified with a QIAquick PCR purification column, the purified solution was concentrated at 40°C for 1 h, and the concentrated labeled DNA was redissolved wit...
Embodiment 3
[0058]According to the conditions described in Example 2, Eucommia ulmoides (Eucommiaulmoides) was subjected to the same treatment, hybridization and detection, and obtained 6 hybridization points with an SNR value greater than 2, and the fingerprints of 4 repetitions were as follows: figure 2 shown.
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