Construction and characterization of a high-quality cDNA library of Cymbidium faberi suitable for yeast one- and two-hybrid assays

Abstract

Background: Cymbidium faberi is one of the oldest cultivars of oriental orchids, with an elegant flower fragrance. In order to investigate the molecular mechanism and the functions of related proteins in the methyl jasmonate (MeJA) signaling pathway, one of the main components of flower fragrance in C. faberi, yeast one-and two-hybrid three-frame cDNA libraries were constructed. Results: In this study, a modified cDNA library used for yeast one-and two-hybrid screening was successfully constructed, with a recombinant efficiency of 95%. The lengths of inserted fragments ranged from 750~3000 bp, and the library capacity reached 6 × 10 9 CFU/ μg of cDNA insert, which was suitable for the requirements of subsequent screening. Finally, a homologous protein related with pathogenesis was screened out by the bait vector of CfbHLH36, which may participate in the MeJA signaling pathway.Conclusion: The yeast one-and two-hybrid library of C. faberi provides large amounts of useful information for the functional genomics research in C. faberi, and this method could also be applied to other plants to screen DNAprotein and protein-protein interactions.

Construction of Yeast One-hybrid Library and Screening of Transcription Factors Regulating LhMYB12-Lat Expression in Asiatic Hybrid Lilies (Lilium spp.)

“…In general, the vital standards for constructing a high-quality yeast-one hybrid library include the purity, integrity and concentration of mRNA. The recombination e ciency, transformation e ciency, and library capacity, including the integral expression information, must contain at least 1 × 10 6 CFU [34,38]. In this study, the three indexes of the yeast library of Lilium were 98%, 400-2000 bp and 1.6 × 10 6 CFU/mL of cDNA insert, respectively, which ful lled the requirements for further library screening.…”

“…The yeast one-hybrid system is generally used to screen prey protein interactions based on bait DNA promoter sequence and analyze transcriptional regulation controlled by TFs that bind to DNA cis-elements located in the gene promoters [28]. Therefore, candidate prey proteins could be obtained by yeast one-hybrid assay, such as in wheat [29], Arabidopsis [30,31], Populus [32], tobacco [33], Cymbidium [34] etc. Nevertheless, until now, the TFs acting on the LhMYB12-Lat gene promoter that positively/negatively responds to anthocyanin biosynthesis and accumulates in the promoter remains unclear in ower plants.…”

Construction of yeast one-hybrid library and screening of transcription factors regulating LhMYBSPLATTER expression in Asiatic hybrid lilies (Lilium spp.)

“…In general, the vital standards for constructing a high-quality yeast-one hybrid library include the purity, integrity and concentration of mRNA. The recombination efficiency, transformation efficiency, and library capacity, including the integral expression information, must contain at least 1 × 10 6 CFU [ 35 , 39 ]. In this study, the three indexes of the yeast library of Lilium were 98%, 400–2000 bp and 1.6 × 10 6 CFU/mL of cDNA insert, respectively, which fulfilled the requirements for further library screening.…”

“…By screening a high-quality library of interactions between target prey proteins and bait plasmid links in regulatory networks enables fast and effective identification in higher organisms [ 31 , 35 , 40 , 41 ]. In this study, we successfully constructed a high-quality cDNA library of LhMYBSPLATTER gene promoter that can be used for yeast one-hybrid assays, providing strong evidences that unknown proteins with functional identification of regulating anthocyanin biosynthesis in Asiatic hybrid lily.…”

“…The yeast one-hybrid system is generally used to screen prey protein interactions based on bait DNA promoter sequence and analyze transcriptional regulation controlled by TFs that bind to DNA cis -elements located in the gene promoters [ 29 ]. Therefore, candidate prey proteins could be obtained by yeast one-hybrid assay, such as in wheat [ 30 ], Arabidopsis [ 31 , 32 ], Populus [ 33 ], tobacco [ 34 ], Cymbidium [ 35 ] etc. Nevertheless, until now, the TFs acting on the LhMYBSPLATTER gene promoter that positively/negatively responds to anthocyanin biosynthesis and accumulation remains unclear in flower plants.…”