Glutathione S-transferase gene LrGSTU3 of lilium regale and application thereof
A lily glutathione and transferase technology, applied in transferase, application, genetic engineering, etc., can solve the problems of metalloenzyme activity reduction, cell membrane permeability change, etc., to shorten the breeding cycle and reduce the use and operation simple effect
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Embodiment 1
[0021] Example 1: LrGSTU3 Full-length cDNA cloning and sequence analysis
[0022] Lilium Minjiang was inoculated with Fusarium oxysporum, and total RNA was extracted from the roots 12 hours after inoculation. The treated root of Lilium Minjiang was ground into powder with liquid nitrogen, then transferred to a centrifuge tube, and total RNA was extracted by guanidine isothiocyanate method. 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), add DEPC water to make up the reaction volume to 14.5 μL; after mixing, heat and denature at 70°C for 5 min, then quickly cool on ice for 5 min, then add 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
[0025] Embodiment 2: plant overexpression vector construction
[0026] Use the SanPrep column type plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert LrGSTU3 coli plasmid pMD18-T- LrGSTU3 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- LrGSTU3 and pCAMBIA2300S for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pMD18-T- LrGSTU3 and pCAMBIA2300S plasmid, add 10 μL 10×K buffer, 5 μL Eco RI, 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 LrGSTU3 The fragment and t...
Embodiment 3
[0029] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants
[0030] The transgenic recipient in this experiment was tobacco ( Nicotiana tabacum L.), the 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.
[0031] Take out the stored pCAMBIA2300S-containing pCAMBIA2300S- LrGSTU3 For the Agrobacterium LBA4404 strain of the plasmid, take 20 uL and inoculate it into 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / L rifampicin, and culture at 28°C until the medium is 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 Agrobacte...
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