浙江红山茶CcFAD2基因克隆及其在籽仁发育中的表达分析

Cloning of CcFAD2 gene from Camellia chekiangoleosa and analysis on its expression during kernel development

基于浙江红山茶(Camellia chekiangoleosa Hu)转录组数据,克隆得到浙江红山茶CcFAD2基因。CcFAD2基因全长1482 bp,开放阅读框长度为1149 bp,编码383个氨基酸。CcFAD2蛋白的理论相对分子质量为44240,理论等电点为pI 8.43,具有4个跨膜结构域,无信号肽,亚细胞定位于内质网膜。CcFAD2蛋白二级结构包含44.39%的α螺旋、11.49%的延伸链、3.92%的β折叠和40.21%的无规卷曲。CcFAD2基因编码的氨基酸序列具有3个保守的组氨酸簇,分别为HECGHH、HRRHH和HVAHH。同源性比对和系统进化树分析结果显示:浙江红山茶CcFAD2蛋白与其他油料植物FAD2蛋白存在高度的保守性,浙江红山茶与油茶(Camellia oleifera Abel.)先聚为一亚组,再与油橄榄(Olea europaea Linn.)聚为一组,明显区别于草本油料植物。实时荧光定量PCR结果显示:CcFAD2基因在整个籽仁发育过程均有表达,其中7月14日(籽仁形成初期)的相对表达量显著高于其他时期。随着浙江红山茶籽仁的发育,籽仁中油酸相对含量在籽仁形成初期最低,之后维持在较高的水平;而亚油酸相对含量在籽仁形成初期最高,之后维持在较低的水平。上述研究结果显示:浙江红山茶籽仁发育过程中,CcFAD2基因相对表达量与亚油酸相对含量的变化趋势一致,推测该基因在浙江红山茶籽仁发育过程中具有调控功能。

CcFAD2 gene from Camellia chekiangoleosa Hu was cloned based on its transcriptome data.The full-length of CcFAD2 gene is 1482 bp,and the length of open reading frame is 1149 bp,which encodes 383 amino acids.The theoretical relative molecular mass of CcFAD2 protein is 44240 and its theoretical isoelectric point is pI 8.43.It has 4 transmembrane domains and no signal peptide,and its subcellular localization is in the endoplasmic reticulum membrane.The secondary structure of CcFAD2 protein includes 44.39%ofα-helix,11.49%of extended strand,3.92%ofβ-turn,and 40.21%of random coil.The amino acid sequence encoded by CcFAD2 gene has 3 conservative histidine clusters,which are HECGHH,HRRHH,and HVAHH,respectively.The homology alignment and phylogenetic tree analysis results show that CcFAD2 protein from C.chekiangoleosa is highly conserved with FAD2 proteins from other oil plants,and C.chekiangoleosa and Camellia oleifera Abel.are clustered into one subgroup first,and then clustered into one group with Olea europaea Linn.,which is evidently different from herbaceous oil plants.The real-time fluorescence quantitative PCR result shows that CcFAD2 gene is expressed throughout the development process of kernel,in which,its relative expression is significantly higher in July 14(early development stage of kernel)than that in other stages.With the development of C.chekiangoleosa kernel,relative content of oleic acid in kernel is the lowest in early development stage of kernel and maintains at a relatively high level later,while relative content of linoleic acid is the highest in early development stage of kernel and maintains at a relatively low level later.It is suggested that during the development process of C.chekiangoleosa kernel,the variation tendency of relative expression of CcFAD2 gene is consistent with that of relative content of linoleic acid,indicating that this gene may have a regulatory function in the development process of C.chekiangoleosa kernel.