Red skinned pear polygalacturonase-inhibiting protein gene (PpPGIP) and application
A technology of polygalactose and protein inhibition, which is applied in the field of molecular biology and genetic engineering, can solve problems such as fungal diseases that cannot be fundamentally solved, reduce the use of disease-resistant agents, reduce environmental pollution, and broaden market application prospects Effect
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[0024] 1. PpPGIP full-length gene cloning and sequence analysis
[0025] 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.
[0026] Using the synthesized first-st...
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