Pseudo-ginseng Dirigent-like protein gene PnDIR1 and application thereof
A protein and gene technology, which is applied in the Panax notoginseng Dirigent similar protein gene PnDIR1 and its application field, can solve the problems of long cycle, difficult to obtain resistance source, and easy loss of resistance of resistant varieties, so as to shorten the breeding cycle and broad market application prospects , cost-saving effect
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Embodiment 1
[0022] Example 1: PnDIR1 Full-length cDNA cloning and sequence analysis
[0023] 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 The total RNA was extracted by the method; M-MLV reverse transcriptase (promega) was used to synthesize the first strand of cDNA using the 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 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 minutes, then quickly cool on ice for 5 minutes, then add 4 μL of 5×First-stand buffer in sequence , 0.5 μL RNasin (200 U), 1 μL M-MLV (200 U), mix well and centrifuge briefly, incubate at 42°C for 1.5 h, ta...
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 PnDIR1 coli plasmid pGEM-T- PnDIR1 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 Xba I and Eco RI respectively for plasmid pGEM-T- PnDIR1 and pCAMBIA2300S for double enzyme digestion (100 μL system), the reaction system and operation process are as follows: take 20 μL pGEM-T- PnDIR1 and pCAMBIA2300S plasmid, add 10 μL 10×H buffer, 5 μL Eco RI, 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 PnDIR1 The fragments and the large fragment of the pCAMBIA2300s vector were gel-recovered separately, and 1 μL o...
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 5-8 days, transfer to light incubator after germination (25°C, 16h / d light), Subculture once a month with MS medium.
[0032] Take out the stored pCAMBIA2300S-containing pCAMBIA2300S- PnDIR1 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 solid me...
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