Lilium regale WRKY transcription factor gene LrWRKY2 and application thereof
A technology of transcription factors and genes, applied in application, genetic engineering, plant genetic improvement, etc., can solve problems affecting the yield and quality of lily cut flowers, decline in bulb quality, scale rot and fall off, etc., and achieve broad market application prospects and breeding cycle Effect of shortening, reducing usage
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[0019] Example 1: LrWRKY2 Full-length gene cloning and sequence analysis
[0020] Minjiang lily was inoculated with Fusarium oxysporum, and total RNA was extracted from the roots 24 h after inoculation. The inoculated roots of Minjiang lily were ground into powder with liquid nitrogen, then transferred to a centrifuge tube, and total RNA was extracted by guanidine isothiocyanate method. RNA, reverse transcriptase M-MLV (promega) was used to synthesize the first strand of cDNA with total RNA as template. The reaction system and operation process were as follows: take 5 μg total RNA, add 50 ng oligo (dT), 2 μL dNTP (2.5 μg) in turn. mM each) and DEPC water to a reaction volume of 14.5 μL; after mixing, heat denaturation at 70°C for 5 min, then quickly cool on ice for 5 min, then add 4 μL 5×First-standbuffer, 0.5 μL RNasin (200U) in sequence , 1 μL M-MLV (200U), mix well and centrifuge for a short time, incubate at 42°C for 1.5 h, take out and heat at 70°C for 10 min to terminat...
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[0023] Example 2: Plant overexpression vector construction
[0024] The insert was extracted using the SanPrep column plasmid DNA mini-extraction kit (Shanghai Shenggong). LrWRKY2 The E. coli plasmid pGEM-T- LrWRKY2 And the plasmid of the plant expression vector pCAMBIA2300s, take 1 μL for agarose gel electrophoresis to test the integrity and concentration of the extracted plasmid; use restriction endonuclease Bam HI (TaKaRa) and Xba I(TaKaRa) was applied to the plasmid pGEM-T- LrWRKY2 Double-enzyme digestion with pCAMBIA2300s (100 μL system), the reaction system and operation process are: take 20 μL pGEM-T- LrWRKY2 and pCAMBIA2300s plasmid, followed by adding 10 μL 10×K buffer, 4 μL Bam HI, 6 μL Xba 1. 60 μL ddH 2 O, centrifuge for a short time after mixing, and place at 37°C for overnight reaction; spot all the digestion products on agarose gel for electrophoresis, and then analyze LrWRKY2 The fragment and the large fragment of the pCAMBIA2300s vector were gel-rec...
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[0027] Example 3: Agrobacterium-mediated plant genetic transformation and screening of transgenic plants
[0028] The transgenic receptor in this experiment was tobacco. Tobacco seeds were 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, sow on 1 / 2 MS medium, cultivate in dark at 28 °C for 6 d, transfer to a light incubator (25 °C, 16 h / d light) after germination, and then transfer to a light incubator (25 °C, 16 h / d light). Subculture once a month with 1 / 2 MS medium.
[0029] Remove the stored pCAMBIA2300s-containing pCAMBIA2300s- LrWRKY2 The plasmid Agrobacterium LBA4404 strain was inoculated into 5 mL of LB liquid medium containing 50 mg / L Km and 20 mg / L rifampicin, 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 cultivate at 28 °C for 48 h; then scrape off an appropriate amount o...
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