A Minjiang lily wrky transcription factor gene lrwrky11 and its application
A technology of transcription factor and gene, which is applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of bulb quality decline, affecting the yield and quality of lily cut flowers, scale rot and falling off, etc.
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Embodiment 1
[0020] Example 1: LrWRKY11 Full-length gene cloning and sequence analysis
[0021] The roots of lily were inoculated with Fusarium oxysporum, total RNA was extracted from the root 24 h after inoculation, the treated root of lily was ground into powder with liquid nitrogen, then transferred into a centrifuge tube, and extracted by guanidine isothiocyanate method Total RNA: Use M-MLV reverse transcriptase (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 in sequence (2.5mM each), make up the reaction volume to 14.5 μL with DEPC water; after mixing, heat denaturation at 70°C for 5 min, then rapidly 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; store at -20°C...
Embodiment 2
[0024] Embodiment 2: plant overexpression vector construction
[0025] Use the SanPrep column plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert LrWRKY11 coli plasmid pGEM-T- LrWRKY11 As well as the plasmid of the plant expression vector pCAMBIA2300S, take 1 μL for agarose gel electrophoresis to detect the integrity and concentration of the extracted plasmid; Bam HI and Xba I respectively against the plasmid pGEM-T- LrWRKY11and pCAMBIA2300S for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pGEM-T- LrWRKY11 and pCAMBIA2300S plasmid, add 10 μL 10×K buffer, 5 μL Bam HI, 5 μL Xba I. 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 the LrWRKY11 The fragments and the large fragments of the pCAMBIA2300S vector were gel-recovered separately, and 1 μL of the reco...
Embodiment 3
[0028] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants
[0029] The transgenic recipient in this experiment was tobacco ( Nicotiana tabacum ), the tobacco seeds were soaked in 75% alcohol for 30s, washed with sterile water and washed with 0.1% HgCl 2 Soak for 8 min, then wash several times with sterile water, sow on 1 / 2MS medium, culture in dark at 28°C for 5-8d, transfer to light incubator (25°C, 16h / d light) after germination, and then Subculture once a month with 1 / 2 MS medium.
[0030] Take out the stored pCAMBIA2300S-containing pCAMBIA2300S- LrWRKY11 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 soli...
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