Application of lilium regale germin protein gene LrGLP1
A germin and protein technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of inability to completely control fungal diseases, harm to human and animal life and health, long cycle, etc., achieve broad market application prospects, shorten the breeding cycle, Simple to use effects
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Embodiment 1
[0022] Example 1: LrGLP1 Full-length cDNA cloning and sequence analysis
[0023] Lilium Minjiang was inoculated with Fusarium oxysporum, total RNA was extracted from the roots 12 hours after inoculation, the treated roots of Lily Minjiang were ground into powder with liquid nitrogen, then transferred to a centrifuge tube, and extracted by guanidine isothiocyanate method total RNA. Use reverse transcriptase M-MLV (promega) to synthesize the first strand of cDNA using total RNA as a template. The reaction system and operation process are as follows: take 5 μg total RNA, add 50 ng oligo (dT), 2 μL dNTP Mix (2.5mM Each), the reaction volume was made up to 14.5 μL with DEPC water; after mixing, heated and denatured at 70°C for 5 min, then rapidly cooled on ice for 5 min, then added 4 μL 5×First-stand buffer, 0.5 μL RNasin ( 200U), 1 μL M-MLV (200U), mix well and centrifuge briefly, incubate at 42°C for 1.5 h, take it out and heat at 70°C for 10 min to terminate the reaction. Aft...
Embodiment 2
[0026] Embodiment 2: plant overexpression vector construction
[0027] Use the SanPrep column type plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert LrGLP1 coli plasmid pMD18-T- LrGLP1 As well as the plant expression vector pCAMBIA2300S plasmid, 1 μL was used for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid. restriction endonuclease Eco RI (TaKaRa) and Bam HI (TaKaRa) against plasmid pMD18-T- LrGLP1and pCAMBIA2300S for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pMD18-T- LrGLP1 and pCAMBIA2300S plasmid, add 10 μL 10×K buffer, 5 μL EcoRI , 5 μL Bam HI, 60 μL ddH 2 O, after mixing, centrifuge for a short time, and place at 37°C for overnight reaction. All digested products were subjected to agarose gel electrophoresis, and then SanPrep Column DNA Gel Extraction Kit (Shanghai Shenggong) was used for LrGLP1 The fragments and the la...
Embodiment 3
[0030] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants
[0031] The transgenic recipient in this experiment was tobacco ( Nicotiana tabacum L.). Tobacco seeds were soaked in 75% alcohol for 30 s, washed with sterile water and washed with 0.1% HgCl 2 Soak for 8 minutes, then wash several times with sterile water, sow on 1 / 2 MS medium, culture in dark at 28°C for 5-8 days, transfer to light incubator after germination (25°C, 16h / d light), Subculture once a month with MS medium.
[0032] Take out the stored pCAMBIA2300S-containing pCAMBIA2300S- LrGLP1 Agrobacterium LBA4404 strain of the plasmid was inoculated in 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / L rifampin in 20 μL, and cultured at 28°C until the medium was turbid. Pipette 1 mL of turbid bacterial solution onto LB solid medium containing 50 mg / L Km, and incubate at 28°C for 48 h. Then scrape off an appropriate amount of Agrobacterium on LB solid me...
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