Thaumatin-like protein gene PpTLP from pyrus pyrifolia nakai with antifungal activity and application
A sweet protein-like, anti-fungal technology, applied in the fields of molecular biology and genetic engineering, can solve the problems of endangering human and animal health, high chemical pesticide residues, long cycle, etc., achieve broad market application prospects, reduce the use of disease-resistant agents, and breed short cycle effect
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[0023] 1) PpTLP full-length gene cloning and sequence analysis
[0024] Grind the red peel of the torched pear into powder with liquid nitrogen, transfer it to a centrifuge tube, and extract total RNA by the guanidine isothiocyanate method. Use reverse transcriptase M-MLV (Tiangen) 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 50ng oligo(dT)15, 2μL dNTP (2.5mM each) in sequence ,ddH 2 O (RNase-free) to a reaction volume of 13.5 μL; 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 (200 U), 1 μL M- MLV (200U), 1 μL DTT (0.1M), mix well and centrifuge for a short time, incubate at 42°C for 1.5h, take it out and heat at 95°C for 5min to terminate the reaction. After the first strand of cDNA was synthesized, it was stored at -20°C for future use.
[0025] Using the synthesized first-stra...
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