Abstract:
Objective A yeast cDNA library was constructed using the leaves from a cold tolerant Amur grape plant under low-temperature stress to facilitate the study of the resistance mechanism.
Method As a well-known frost resistant Vitis amurensis Rupr., V. amuerensis cv. Zuoshan-1 was cultured in a pot experiment for a total RNA extraction from the seedling leaves after 0, 2, 4, 8, 12, and 24 h at 4 °C. The extract was then reverse transcribed into cDNA using the SMART technology to be purified. Short fragments were removed and ligated into pGADT7 3-frame plasmid vector to establish a primary cDNA library. After amplification, plasmid was extracted and transformed into yeast Y187 for the library construction, followed by a qualitative verification.
Result The capacities of the 3 reading frame primary libraries were tested to be 1.7×106 cfu, 2.0×106 cfu, and 1.9×106 cfu with a recombination rate of 100%. The lengths of the inserted fragments were largely in the 500-2 000 bp region. The electrophoretic detected and sequenced encoded proteins corresponding to the inserted fragments were abundant and highly polymorphic. The titer of the finally obtained Y187 yeast library was approximately 4.0×108 cfu·mL−1.
Conclusion A high quality yeast cDNA library using Amur grape leaves under low temperature was successfully constructed for future studies on the interacting proteins under cold stress that could induce injury impairing the growth, development, fruit quality, and yield of grape vines.
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