Cloning and expression analysis of LEC1 gene from Xanthoceras sorbifolia embryos

ObjectiveXanthoceras sorbifolia, belonging to the family Sapindaceae, is one of the most valuable oil trees widely distributed in northern China. Its seeds contain a large amount of oil that is high-quality raw material for food and biodiesel purposes. This study aims to clone Xanthoceras sorbifolia Leafy Cotyledon 1gene (XsLEC1) and analyze its sequence and expression pattern.

MethodXsLEC1 gene was cloned by RT-PCR and RACE methods from developing X. sorbifolia embryos. Physicochemical properties and transmembrane regions of the deduced XsLEC1 protein were predicted via Protparam and TMHMM2.0 programs, respectively. Subcellular localization and function of XsLEC1 protein were predicted by ProtComp9.0 and Motif Scan programs, respectively. Multiple sequence alignment was carried out using BioEdit software and phylogenetic analysis was achieved by MEGA7 software. Expression pattern of XsLEC1 was analyzed by RT-PCR and quantitative real-time PCR.

ResultXsLEC1 cDNA was 1 035 bp in length, containing a 690 bp ORF (GenBank accession numbers: MF616360) and encoding a putative protein with 229 amino acids. The deduced amino acid sequence of XsLEC1 contained a conserved B domain of the HAP3 subunit. The B domain contained three α-helices (α1, α2, α3) and two loops (L1, L2) in the histone fold motif. The predicted XsLEC1 was a stable hydrophilic protein without signal peptide and transmembrane regions. The protein was located in the nucleus and contained many phosphorylation sites. The secondary structure of XsLEC1 protein was mainly consisted of alpha helix and random coil. Phylogenetic analysis showed that XsLEC1 was the closest to DlLEC1, followed by JcLEC1 and PtLEC1. The RT-PCR revealed that XsLEC1 was not expressed in roots, stems, leaves, and petals, but highly expressed in the developing embryos. Quantitative real-time PCR indicated that XsLEC1 had temporal expression pattern in developing X. sorbifolia embryos. The XsLEC1 expression was higher in early embryo development (33, 40 and 47 days after anthesis) than in late embryo development (54, 61 and 68 days after anthesis). With the embryo maturation, the XsLEC1 expression was very low at 75 days after anthesis and no transcript was detected at 81 days after anthesis.

ConclusionThe cloning and expression analysis of XsLEC1 gene provide an important foundation to further study the function of XsLEC1 and are of practical significance for improving the quality of the X. sorbifolia oil.