Yeast Two-hybrid cDNA Library Constructed from Vitis davidii Fex Pericarps Infected by Grape Ripe Rot Pathogen, Colletotrichum viniferum

Abstract

Objective A yeast two-hybrid cDNA library on Colletotrichum viniferum-infected pericarps of the wild spine grape (Vitis davidii Foex), a cultivar highly resistant to the serious grape ripe rot in southern China, was constructed to facilitate studies on the molecular mechanism of the disease resistance in the plants. Method The total RNA was extracted from the pericarps of V. davidii accession, Fu'an, 1, 3, and 7d after C. viniferum inoculation and reverse-transcribed into cDNA using the SMART method. The cDNA was purified by an assay kit into a double stranded cDNA to be digested. Then, the short fragments were removed to obtain a high-quality cDNA for the subsequent cloning into a pGADT7 3-frame plasmid vector and purifying for the establishment of a primary cDNA library. After amplification, the plasmid was extracted and transformed into Yeast Y187 to produce the amplified library for final identification. Result The primary cDNA library had a capacity of approximately 5.2×106 cfu with a recombination rate of approximately 97.92% and the inserted fragments of good polymorphism in the lengths ranging from 400 to 2 000 bp. The harvested Y187 yeast library titer was about 6.0×107 cfu·mL−1. Conclusion The constructed cDNA library from C. viniferum-infected grape pericarps was adequate for yeast two-hybrid screening. It would materially aid the studies on the interacting proteins in grape plants infected by the pathogen.