Panax notoginseng reverse osmosis protein gene PnOLP1 and application

[0019] Example 1: PnOLP1 Full-length gene cloning and sequence analysis

[0020] The roots of Panax notoginseng were inoculated with Fusarium solani rot, total RNA was extracted from the roots 12 h after inoculation, the treated roots of Panax notoginseng were ground into powder with liquid nitrogen, and then transferred to a centrifuge tube, and guanidine isothiocyanate was used to Total RNA was extracted using the method; M-MLV reverse transcriptase (promega) was used to synthesize the first strand of cDNA using total RNA as a template. The reaction system and operation process were as follows: take 5 μg Total RNA, add 50 ng oligo (dT), 2 μL dNTP in sequence Mix (2.5 mM each), make up the reaction volume to 14.5 μL with DEPC water; 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

[0023] Embodiment 2: plant overexpression vector construction

[0024] Use the SanPrep column plasmid DNA mini-extraction kit (Shanghai Sangong) to extract the insert PnOLP1 coli plasmid pGEM-T- PnOLP1 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 Eco RI respectively for plasmid pGEM-T- PnOLP1 and pCAMBIA2300S for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pGEM-T- PnOLP1 and pCAMBIA2300S plasmid, add 10μL 10×H buffer, 5μL Eco RI, 5 μL Bam HI, 60 μL ddH 2 O, after mixing, centrifuge for a short time, and place it at 37°C for overnight reaction; perform agarose gel electrophoresis on all digested products, and then use the kit to analyze PnOLP1 The fragments and the large fragments of the pCAMBIA2300S vector were gel-recovered separately, and 1 μL of the recovered

[0027] Example 3: Plant genetic transformation mediated by Agrobacterium and screening of transgenic plants

[0028] 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 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 after germination (25°C, 16h / d light), and then Subculture once a month with MS medium.

[0029] Take out the pCAMBIA2300s- containing pCAMBIA2300s-PnOLP1 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 the Agrobacterium on LB solid medium and inoculate