Application of Arabidopsis sec14p-like gene in fluorescent labeling of lipid droplets in plant cells
A sec14p-like-r, sec14p-like-f technology, applied in the field of genetic engineering, can solve the problems of expensive probes, obstacles to the development and progress of lipid droplet research, and high cost, and achieve the effect of wide application prospects
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Embodiment 1
[0038] Example 1 Cloning and vector construction of Sec14p-like gene
[0039] (1) PCR amplification of Sec14p-like gene
[0040] The vector was constructed in GATEWAY mode, and primers were designed according to the published Arabidopsis thaliana gene AT1G14820 sequence. The size of the amplified product was 817bp, of which the gene coding region was 756bp, and the stop codon at the end of the coding region for the expression of the fusion protein had been removed.
[0041] Sec14p-like-F: ggggacaagtttgtacaaaaaagcaggcttcATGGAGGAAAGCCAAG
[0042] Sec14p-like-R:ggggaccactttgtacaagaaagctgggtcAACATTATTGTTTGTTAGAG
[0043] Arabidopsis RNA was extracted, reversed into cDNA, and PCR amplified using the above primers. The base sequences of the primer pair are respectively shown in SEQ ID NO.3 and SEQ ID NO.4.
[0044] (2) Recovery of target fragments and construction of intermediate vectors
[0045] The target DNA fragment was recovered by agarose gel electrophoresis ( figure 1 ...
Embodiment 2
[0051] Example 2: Recombinant vectors pEarleygate101-Sec14p-like-YFP and pEarleygate103- Tobacco transient transformation and lipid droplet labeling analysis of Sec14p-like-GFP
[0052] (1) Transform the recombinant vector into Agrobacterium
[0053] The recombinant vector pEarleygate101-Sec14p-like-YFP was transformed into Agrobacterium EHA105 by freeze-thaw method, and the single clone of Agrobacterium on the plate was picked and inoculated into 1ml liquid resistant LB medium (streptomycin+rifampicin+ kanamycin) and cultured in a shaker at 28°C for 24 hours.
[0054] (2) Transiently transformed tobacco
[0055] The bacterial solution was transferred to 10ml liquid medium (containing 10mM MES and 40uM AS), and cultured in a shaker at 28°C for 16 hours. Centrifuge the bacterial liquid to collect the bacterial cells, and use 10mM MgCl 2 Resuspend bacteria in solution to OD 600 =1, finally add AS to a final concentration of 200uM and let stand for 3 hours. Get the three...
Embodiment 3
[0060] Example 3: The recombinant vector pEarleygate101-Sec14p-like-YFP stably transforms Arabidopsis thaliana and lipid droplet markers note analysis
[0061] (1) Arabidopsis planting and transformation
[0062] Sow Arabidopsis Col-0 seeds in the culture soil, transplant the seedlings into large pots after seven days, and manage and cultivate them normally in the greenhouse with a temperature of 20-22°C, a relative humidity of 50-70%, and a light intensity of 100-120 μmol / (m 2 .s), 16h light and 8h dark.
[0063] Take the Agrobacterium EHA105 transformed with the recombinant vector pEarleygate101-Sec14p-like-YFP in Example 2, inoculate it into liquid LB medium, and culture it overnight at 28° C. and 200 rpm. The bacteria liquid was collected by centrifugation at 4000rpm for 10min. Resuspend Agrobacterium with transformation medium to OD 600 Approximately equal to 1.0. Pour the bacterial solution into a petri dish to prepare for flower dipping infection. Immerse all A...
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