Promoter cloning of PuLOX2S gene from “Nanguo” pears and screening of transcription factors by Y1H technique

Our previous study on differential proteome and transcriptome of refrigerated "Nanguo" pears found that the PuLOX2S gene was very active in the LOX pathway of aroma synthesis, but the regulation of expression behavior of the gene and how to mediate the aroma synthesis were still unknown. Partial genome sequences of PuLOX2S were cloned, and its promoter was analyzed by Tail-PCR. The PuLOX2S promoter sequences of 610 bp were isolated and identified using Plant CARE, which were composed of cis-acting elements, such as ABRE, AE-box, ARE, CAAT-box, Box 4, TCCC-motif, CAT-box, CGTCA-motif, G-Box, TATA-box, TCA-element, TGA-element, and TGACG-motif. The Y1H technology was used to determine whether proteins interacted with PuLOX2S based on the pGADT7-Chinese white pear cDNA library. The Y1H results were shown that 52 proteins could interact with the PuLOX2S promoter, which was compared with sequences in the GenBank database. The three genes PuERF12, PuMYB44, and PuRF2a were the candidate transcription factors of PuLOX2S and PuCDPK10 played an important role in the gene expression in Nanguo pears. Therefore, the results of this study supply important information for revealing new function of PuLOX2S and the regulation mechanism of expression behavior of the gene. It provides new ideas for the regulation of aroma synthesis in Nanguo pears. PRACTICAL APPLICATIONS: The gene PuLOX2S was very active in the LOX pathway of aroma synthesis, but the regulation of expression behavior of the gene and how to mediate the aroma synthesis were still unknown. We have successfully cloned the partial sequence of the gene and the 610 bp promoter sequence upstream of PuLOX2S and analyzed the structure of cis-acting elements. There are 52 proteins that interact with the PuLOX2S promoter revealed by the Y1H technique. Three transcription factors among the proteins can regulate the level of PuLOX2S expression, which provides new ideas for the regulation of aroma synthesis in "Nanguo" pears. Moreover, the study results could supply scientific information for the quality improvement and genetic modification of Nanguo pears.