Construction of Yeast Two-Hybrid Three-Frame cDNA Library and Screening of Interacting Proteins in FWL1 Membrane System of Pear Young Fruit

【Objective】 Screening of proteins that interact with FWL1 provides a basis of in-depth exploration of the mechanism of FWL1 gene regulation of fruit size. 【Methods】 Using ‘Duli’ pear, Korla fragrant pear, and Yali pear and Zaomeixiang were used as materials, to extract the mixed RNA of pulp tissue in the critical period of fruit cell division of four pear varieties was extracted. And then a yeast two-hybrid triple-frame cDNA library of pear young fruit FWL1 membrane system was constructed, and the quality of the library was identified. After that, The the bait vector was constructed and transformed into yeast, and the proteins that interacted with FWL1 were screened. The positive yeast clones that were initially screened were sequenced and Blast alignment was performed in NCBI to determine the candidate interacting proteins. 【Results】 The library capacity is was about 3×10⁷ CFU, greater than 1×10⁷ CFU, the average insert fragment is was greater than 1,000 bp, and the positive rate is was ≧98%. After testing the bait vector had no self-activating function, and 272 proteins interacting with FWL1 were screened out by co-transformation method. 【Conclusion】 After testing, the yeast two-hybrid three-frame cDNA library of the pear young fruit membrane system meets the basic conditions of the yeast two-hybrid library and is suitable for later screening of interacting proteins. The screened 272 interacting proteins were are expressed differently during fruit development, among which collagen and calcium-binding EGF domain-containing protein 1 and metallothionein-like protein may be related to pear fruit size development.