Application of the Germin-Like Protein Gene lrglp1 of Minjiang Lily
A germin and protein technology, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of incomplete control of fungal diseases, human and animal life and health hazards, long cycle, etc., achieve broad market application prospects, shorten the breeding cycle, Simple operation effect
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Embodiment 1
[0022] Example 1: LrGLP1 Full-length cDNA cloning and sequence analysis
[0023] oxysporum was used to inoculate Lilium Minjiang, 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 the total RNA was extracted by guanidine isothiocyanate method. 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 ngoligo (dT) and 2 μL dNTPMix (2.5 mMeach) in sequence, and dilute the reaction volume with DEPC water. Make up to 14.5 μL; after mixing, heat denaturation at 70°C for 5 minutes, then quickly cool on ice for 5 minutes, then add 4 μL 5×First-standbuffer, 0.5 μL RNasin (200 U), 1 μL M-MLV (200 U), mix well and briefly centrifuge , in a warm bath at 42°C for 1.5h, and after taking it out, heat it at 7...
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- LrGLP1 and 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 10μL 10×Kbuffer, 5μL EcoRI , 5μL 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 recovery kit (Shanghai Shenggong) was used to LrGLP1 The fragments and the large frag...
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 5-8d, transfer to light incubator (25°C, 16h / d light) after germination, and then monthly Subculture once with MS medium.
[0032] Take out the stored pCAMBIA2300S-containing pCAMBIA2300S- LrGLP1 For the Agrobacterium LBA4404 strain of the plasmid, take 20 μL 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 1mL of turbid bacterial solution onto LB solid medium containing 50mg / LKm, and incubate at 28°C for 48h. Then scrape off an appropriate amount of Agrobacterium...
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