文冠果LEC1基因的克隆及表达分析

Cloning and expression analysis of LEC1 gene from Xanthoceras sorbifolia embryos

【目的】文冠果隶属无患子科,是我国北方重要的油料树种,种子含油量高,是制备食用油、生物柴油等的优质原料。本研究旨在克隆文冠果Leafy Cotyledon 1(XsLEC1)基因,进行序列及表达模式的分析。【方法】以文冠果种胚为试材,利用RT-PCR和RACE技术克隆文冠果LEC1基因。采用Protparam及TMHMM2.0软件预测XsLEC1蛋白的理化性质和跨膜结构域,Prot Comp9.0及Motif Scan软件预测XsLEC1蛋白的亚细胞定位及蛋白功能,BioEdit及MEGA7软件分析XsLEC1蛋白的多重序列比对和进化关系。运用RT-PCR和实时荧光定量PCR分析XsLEC1基因的表达模式。【结果】XsLEC1 cDNA全长1 035 bp,包含一个长度为690 bp的开放阅读框(GenBank号为MF616360),可编码229个氨基酸。推导的氨基酸序列包含一个HAP3亚基的保守功能域B。该保守功能域内含有组蛋白折叠中的3个α螺旋(α1、α2、α3)和两个短环结构(L1、L2)。在线预测该蛋白为稳定的,亲水性蛋白,无信号肽和跨膜区域,定位于细胞核,存在多个磷酸化位点。二级结构主要由α螺旋和无规则卷组成。进化分析表明,该蛋白序列与同科龙眼亲缘关系最近,其次为麻风树和毛果杨。RT-PCR实验得出,XsLEC1基因在文冠果的根、茎、叶及花中均无表达,在种子中出现较高表达。实时荧光定量PCR结果进一步显示,XsLEC1基因在种胚中有明显的时序表达特性,在种胚发育的前期(花后33、40、47 d)表达较高,在种胚发育的后期(花后54、61、68 d)表达较低,至花后75 d时仅有微量表达,而在完全成熟的种胚(花后81 d)中未检测到XsLEC1的表达。【结论】文冠果LEC1基因的克隆及表达分析,为以后深入研究XsLEC1的功能奠定了分子基础,同时对生产实践中文冠果油脂品质的改良有一定的实践意义。

[ Objective] Xanthoceras 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 l gene （XsLEC1） and analyze its sequence and expression pattern. [ Method] XsLEC1 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. [ Result] XsLEC1 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 a-helices （otl, or2, c^3） 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 XsLECI protein was mainly consisted of alpha helix and random coil. Phylogenetic analysis showed that XsLEC1 was the closest to D1LEC1, 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. [ Conclusion~ The 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.