Notoginseng class sweet protein gene PnTLP5 and application thereof
A kind of sweet protein and gene technology, applied in the notoginseng sweet protein gene PnTLP5 and its application field, to achieve the effect of saving cost, reducing environmental pollution, and simple operation
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[0019] Example 1: PnTLP 5 Full-length gene cloning and sequence analysis
[0020] Take the three-year-old Panax notoginseng leaves to extract total RNA, grind the Panax notoginseng leaves into powder with liquid nitrogen, then transfer them into a centrifuge tube, use the guanidine isothiocyanate method to extract total RNA, and use reverse transcriptase M-MLV (promega) to Total RNA is used as the template to synthesize the first strand of cDNA. The reaction system and operation process are as follows: Take 5 μg Total RNA, add 50 ng oligo (dT), 2 μL dNTP (2.5 mM each), DEPC water to the reaction volume to 14.5 μL; After mixing, heat and denature at 70°C for 5 minutes and then quickly cool on ice for 5 minutes, then add 4 μL 5×First-stand buffer, 0.5 μL RNasin (200U), 1 μL M-MLV (200U), and mix well. Centrifuge for a short time, warm bath at 42°C for 1.5 h, take it out and heat at 70°C for 10 min to terminate the reaction. After the first strand of cDNA is synthesized, it is sto...
Example Embodiment
[0023] Example 2: Plant overexpression vector construction
[0024] Use SanPrep column plasmid DNA small extraction kit (Shanghai Shenggong) to extract inserts PnTLP5 Escherichia coli plasmid pMD-18T- PnTLP5 And the plasmid of the plant expression vector pCAMBIA2300s, take 1 μL for agarose gel electrophoresis to check the integrity and concentration of the extracted plasmid; use restriction enzymes Bam HI (TaKaRa) and Eco RI (TaKaRa) was used for plasmid pMD-18T- PnTLP5 Double enzyme digestion with pCAMBIA2300s (100 μL system), the reaction system and operation process are: take 20 μL pMD-18T- PnTLP5 And pCAMBIA2300s plasmid, sequentially add 10 μL 10×K buffer, 5 μL Bam HI, 5 μL Eco RI, 60 μL ddH 2 O, centrifuge for a short time after mixing, and place it at 37°C overnight for reaction; spot all digested products on agarose gel for electrophoresis, and then PnTLP5 The fragment and the large fragment of pCAMBIA2300s vector were separately recovered by gel; 1 μL of the recovered ...
Example Embodiment
[0027] Example 3: Agrobacterium-mediated plant genetic transformation and transgenic plant screening
[0028] The transgenic recipient in this experiment is tobacco. Tobacco seeds are soaked in 75% alcohol for 30s, washed with sterile water and then washed with 0.1% HgCl 2 Soak for 8 min, then wash several times with sterile water, sown on 1 / 2 MS medium, cultivate in the dark at 28°C for 6 d, and transfer to a light incubator (25°C, 16h / d light) after germination. Subculture once a month with 1 / 2MS medium.
[0029] Take out the preserved pCAMBIA2300s- PnTLP5 The plasmid Agrobacterium LBA4404 strain was inoculated in 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / L rifampicin, and cultured at 28°C until the medium became turbid. Pipette 1 mL of the turbid bacterial liquid onto the LB solid medium containing 50 mg / L Km, and incubate at 28°C for 48 h; then scrape off the Agrobacterium on the LB solid medium and inoculate it with 20 mg / L acetyl. In the MGL liquid medium of s...
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