The application of triticin-5-o-glucoside in regulating plant resistance to weeds
A technology of ticlavone and glucose, applied in the application field of ticlavone-5-O-glucoside in regulating plant resistance to weeds, can solve problems such as genetic mechanism and weed suppression mechanism are not very clear
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Embodiment 1
[0055] Example 1. Compound purification
[0056] (1) Collect the fresh leaves of rice (indica) about 60 days after sowing, and grind them into powder after drying with a freeze dryer. 8 times the volume of 75% ethanol was added, and ultrasonic extraction was performed three times for 20 min each time.
[0057] (2) Filter the extract, and use a rotary evaporator to concentrate it until there is no alcohol smell, and sequentially add equal volumes of petroleum ether, dichloromethane, and n-butanol to extract 3 times respectively.
[0058] (3) After concentrating the n-butanol phase obtained with a rotary evaporator, a medium and low pressure rapid preparation liquid chromatograph (Biotage Isolera TM Prime) using wet loading for coarse fractionation. The silica gel column packing is YMC*GEL C18 spherical packing with a particle size of 50 μm and a pore size of 12 nm. The weight of the filler used in this example is 100 g. The mobile phase is water (containing 0.1% formic acid...
Embodiment 2
[0065] Example 2, Os07g0503900 gene cloning
[0066] (1) The genomic DNA of the leaves of the japonica rice Zhonghua 11 (Oryza sativa L.ssp.japonica cv.Zhonghua11, ZH11) at the seedling stage was extracted.
[0067] (2) According to the existing Os07g0503900 sequence information on the Rice Genome Annotation Project, primers F1 / R1 containing BamHI and HindШ were used to amplify the CDS sequence of the Os07g0503900 gene containing the restriction site with the ZH11 genomic DNA as the template.
[0068] Primer F1: 5'-ggatccATGGCTCCAGCGATGGCGAG-3';
[0069] Primer R1: 5'-aagcttCTATATGGATGACATGTGGGC-3'.
[0070] (2) ligating the obtained gene fragment to pEASY-T 3 Vector (TransGen Biotech, -T3Cloning Kit), transformed Escherichia coli DH5α competent cells, and screened positive clones using blue-white spots.
[0071] (3) The positive clones were identified by PCR using primers F2 / R2, and the amplified fragment size was 310bp as positive clones. The primers are as follows:
...
Embodiment 3
[0076] Embodiment 3, the application of Os07g0503900 gene
[0077] 1. Construction of prokaryotic expression vector
[0078] (1) The recombinant vector pEASY-T obtained in Example 2 was transformed with restriction enzymes BamHI and HindШ 3 -Os07g0503900 was completely digested, and the expression vector pMAL-c2X (Hua Yue Yang, VECT-570) was digested simultaneously. The digestion system was: 5 μg plasmid, 2.5 μL 10× digestion buffer, 2 μL BamHI, 2 μL HindШ, plus ddH 2 O replenish the reaction system to 50 μL. The restriction enzyme digestion reaction conditions were: 37°C for 4 hours.
[0079] (2) Separate the digested products by agarose electrophoresis, and recover a fragment of about 1.5Kb containing Os07g0503900 and a pMAL-c2X vector fragment of about 6.6Kb, respectively, dissolved in 30 μL of ddH 2 O in.
[0080] (3) The gene fragments obtained in step (2) are respectively ligated with the vector backbone fragments. The ligation reaction system is: 1 μL 10× ligase b...
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